Process of selectively blocking amino functions in aminoglycosides using transition metal salts and intermediates used thereby

ABSTRACT

Selective blocking of some amino groups in a polyamino organic compound having at least one pair of available neighboring hydroxyl and amino groups is effected by first preparing in situ transition metal salt complexes of available neighboring amino and hydroxyl group pairs in said polyamino organic compound, followed by introduction of blocking groups on the non-complexed amino groups and, finally, removing the transition metal cations from the selectively N-blocked polyamino organic compound complex to obtain a polyamino organic compound having selectively blocked amino groups. 
     This process is particularly valuable when carrying out aminocyclitol-aminoglycoside transformations utilizing transition metal salt complexes of cupric acetate, nickel (II) acetate, cobalt (II) acetate or mixtures thereof.

FIELD OF INVENTION

This invention relates to a novel process useful particularly incarrying out aminocyclitol-aminoglycoside transformations, and to novelderivatives produced thereby and their use as intermediates.

More particularly, this invention relates to a process of selectivelyblocking certain amino groups in a polyamino organic compound utilizingas intermediates, novel transition metal salt complexes of availableneighboring hydroxyl and amino group pairs in said polyamino organiccompound. This invention also relates to novel, selectivelyblocked-amine derivatives produced thereby which are useful asintermediates whereby process improvements are effected.

Specifically, this invention relates to a process for preparingselectively blocked amino derivatives of aminocyclitol-aminoglycosides,useful as intermediates in preparing other aminocyclitol-aminoglycosidederivatives having antibacterial activity, which comprises preparing, insitu, transition metal salt complexes of available neighboring amino andhydroxyl functions in said aminocyclitol-aminoglycoside, followed byintroduction of acyl blocking groups on the non-complexed aminofunctions, thence removal of the transition metal salt cations. Thisinvention also includes novel divalent transition metal salt complexesof said aminocyclitol-aminoglycosides as well as novel selectivelyblocked-amine derivatives of said aminocyclitol-aminoglycosides andtheir use as intermediates.

PRIOR ART

When carrying out certain chemical transformations of apolyamino-organic compound, particularly those in the carbohydrate andaminocyclitol-aminoglycoside art, in order to obtain good yields of adesired product, it is necessary to first prepare derivatives of aminofunctions in the molecule with "blocking" or "protecting" groups toprevent the amino functions from entering into competing reactions withthe reagents being employed. Commonly employed blocking groups are acylblocking groups which are easily attached to amino groups and can beremoved after the desired transformation has been completed but whichare stable under the reaction conditions to be employed.

Chemical transformations at a site other than at an amino function in apolyamino-organic compound usually involves the preparation of aper-N-acylated intermediate by reaction of the polyamino-organiccompound with excess acylating reagent, followed by chemicaltransformation of the per-N-acylated intermediate and thence removal ofthe N-acyl protecting groups by methods well known in the art.

Chemical transformations in a polyamino-organic compound wherein thesite of reaction is to be one of the amino functions, are ideallycarried out on intermediates wherein every other amino function isselectively protected by a blocking group; otherwise, mixtures ofvarious mono-N- and poly-N- derivatives are formed which require tediousseparation techniques (usually chromatography or severalchromatographies) to isolate the desired transformation product.However, it is not always possible to prepare the ideal selectivelyblocked intermediate by prior art methods so that, with amino functionsunprotected, the desired transformation product is produced only in lowyields.

When a polyamino-organic compound contains amino functions of variousdegrees of reactivity due either to steric factors and/or to the primaryand/or secondary and/or tertiary nature of the amine groups, it issometimes possible, although in moderate to low yields, to selectivelyprotect some amino functions while leaving other amino functionsunprotected at which a desired reaction may take place. Such proceduresusually involve multistep transformations requiring the isolation andpurification of each intermediate. Frequently, it is impossible toprotect all amino functions other than the one at which a transformationis desired, such as, for example, when two amino functions are of equalreactivity, so that at best only some amino functions are blocked whichminimizes the number of products formed in a given reaction but whichstill will give rise to a mixture of products resulting in poor yieldsof desired products isolatable and purifiable only with difficulty.

By our invention, it is now possible to easily prepare, in good yields,selectively blocked amino derivatives of polyamino organic compounds inwhich at least one amino function has an available neighboring hydroxylgroup, by a one vessel-two-step procedure comprising the reaction in aninert organic solvent of said polyamino organic compound with a divalenttransition metal salt followed by the reaction in situ of the novelpolyamino-organic compound-transition metal salt complex thereby formedwith an acylating agent whereby N-acyl derivatives of the non-complexedamino functions are formed. Upon removal of the transition metal cationfrom the complex, there are produced selectively blocked N-acylderivatives of said polyamino organic compound which are easily isolatedin good yields of high purity product or which can be further reacted insitu prior to isolation and removal of the acyl blocking groups. Byslight modifications in the choice of solvent and/or in the relativemolar quantities of transition metal salt or acylating agent employed,one can regulate which and how many amino functions are N-acylated.

Our invention finds its greatest use in the carbohydrate andaminocyclitol-aminoglycoside art whereby it is now possible to easilyprepare in good yields N-acylated derivatives heretofore impossible tomake or heretofore produced only in small quantities. Additionally, byour process, it is possible to remove the transition metal cation fromthe reaction mixture by precipitation as the sulfide salt and separationby filtration so that the resulting filtrate comprising the selectivelyN-acylated polyamino-organic compound is of sufficient purity andpredictable yield to allow further reactions to be carried out withoutisolation of the N-acylated polyamino intermediate.

Prior to our invention, divalent transition metal salt complexes ofneighboring amino and hydroxyl group pairs were unknown. The prior artpostulates (Bull. Chem. Soc. Japan, Vol. 39, No. 6, 1235-1248 (1966))the existence of cupraammonium complexes between amines on a chiralcenter and adjacent vicinal hydroxyl groups on a chiral center in situin dilute aqueous ammonia solutions, said cupraammonium complexes beingused for optical rotatory studies as an analytical tool in determiningthe absolute spatial relationship between vicinal amino/hydroxyl grouppairs. The prior art cupraammonium complexes of vicinal amino/hydroxylgroup pairs are described as being formed only in water in the presenceof excesses of cupraammonium ion and are used only to measure opticalrotation or circular dichroism.

By our invention, we have discovered that divalent transition metal saltcomplexes (including copper (II) complexes) of neighboringamino/hydroxyl group pairs (wherein the amino and hydroxyl groups may ormay not be vicinal and may or may not be on chiral centers) can beformed and, moreover, can be formed in an organic solvent with a minimalquantity of divalent transition metal ion (e.g. copper (II) ion) withouthaving to use ammonium hydroxide to form a transition metal ammonium ion(e.g. cupra-ammonium ion). By our invention we have also discovered thatthe polyamino-organic compound-transition metal salt complexes of thisinvention will survive under acylating reaction conditions whichtransform a free amino group to an N-acylated derivative thereof and,furthermore, that the transition metal salt complexes of this inventionwill prevent the complexed amino/hydroxyl groups from entering into thereaction. Also, we have discovered that a divalent transition metalcomplex of this invention (e.g. a copper (II) complex) can bedecomplexed by release of the transition metal cation (e.g. a copper(II) cation) and that the polyamino-organic compound can be recoveredintact. By our invention, therefore, we have discovered a novel andsimple method for preparing blocked derivatives in situ of amino groupshaving an available neighboring hydroxyl function not necessarily in thevicinal positions by preparation of a transition metal complex (e.g. acopper (II) complex) thereof which, after reaction of the complexedmolecule with an acylating reagent whereby N-acyl derivatives ofnon-complexed amino functions are prepared, can be converted, byremoving the transition metal cation (e.g. a copper (II) cation), to aselectively N-acylated polyamine compound free of divalent transitionmetal cations, e.g. copper (II).

DESCRIPTION OF THE PROCESS ASPECT OF THE INVENTION

The process sought to be patented resides in the concept of a processfor selectively blocking amino groups with an acyl blocking group, Y, ina polyamino-organic compound, at least one of said amino groups havingan available neighboring hydroxyl group;

which comprises the reaction of said polyamino-organic compound in aninert organic solvent, with a salt of a divalent transition metal cationselected from the group consisting of copper (II), nickel (II), cobalt(II), cadmium (II) or with mixtures thereof, whereby is formed a complexof said polyamino-organic compound between said transition metal saltand said available neighboring amino and hydroxyl group pairs;

followed by the reaction in situ of the resulting polyamino-organiccompound-transition metal salt complex with an amine blocking reagenthaving an acyl blocking group, Y; thence reaction of the resultingpolyamino-organic compound-transition metal salt complex having acylblocking groups, Y, on non-complexed amino groups, with a transitionmetal precipitating reagent or with ammonium hydroxide, wherebytransition metal cation is removed.

In this specification and in the claims the phrases "one of said aminogroups having an available neighboring hydroxyl group" and "availableneighboring amino and hydroxyl group pairs" include amino and hydroxylfunctions which are positioned on adjacent carbon atoms in a cis-vicinalor diequatorial trans-vicinal manner and also includes amino andhydroxyl groups which are not positioned on vicinal carbons but whichare spatially adjacent (i.e. they are close enough to be hydrogenbonded) and available to each other. Further, by the term "available" ismeant that, in addition to being available to each other, to be usefulin the process of this invention, the neighboring amino and hydroxylgroup pairs must be so situated in the polyamino-organic compoundmolecule so as to be sterically available to an approaching reagent suchas a cation of a divalent transition metal. Thus, for example, in a4,6-di-O-(aminoglycosyl)-2-deoxystreptamine having a 2'-amino group suchas in sisomicin, tobramycin, verdamicin and gentamicins C₁, C_(1a) andC₂, although in one of the conformers, the 5-hydroxyl group of the2-deoxy-streptamine moiety is neighboring and available to the 2'-aminogroup, the 5-hydroxyl group is so sterically hindered that a salt of adivalent transition metal such as cupric acetate, cannot get intosufficiently close proximity to the 5-hydroxyl-2'-amino pair to form atransition metal salt complex thereof.

Transition metal salts useful as complexing agents in our processinclude any divalent salt of copper (II), nickel (II), cobalt (II) andcadmium (II). Among those which have strongest complexing activity aredivalent transition metal salts of weak acids, preferably weak organicacids such as benzoic, propionic, and acetic acid. Preferred divalenttransition metal salts include the acetate salt of copper (II), nickel(II), cobalt (II), and cadmium (II) and mixtures thereof. Of particularuse are nickel (II) acetate, copper (II) acetate and cobalt (II)acetate.

In the aminocyclitol-aminoglycoside art, we have found divalenttransition metal halide salts, particularly cupric chloride, to beuseful in improving yields when preparing a 6'-N-acyl derivative of a4,6-di-O-(aminoglycosyl)-2-deoxystreptamine but that, when a3,6'-di-N-acyl or 3,2',6'-tri-N-acyl derivative of the sameaminoglycoside is desired, better results are obtained when a divalenttransition metal acetate, particularly cupric acetate, nickel (II)acetate, cobalt (II) acetate or mixtures thereof, is employed ascomplexing agent.

When preparing selectively blocked N-acyl derivatives ofaminocyclitol-aminoglycosides, divalent transition metal salts of stronginorganic acids, e.g. the sulfate, phosphate and nitrate salts, are lessdesirable as complexing agents than the corresponding salts of weakorganic acids such as the acetate, benzoate and propionate salts.

In our process, in order to obtain strongly bound complexes of theavailable neighboring amino and hydroxyl group pairs, and thus obtainselectivity when N-acylating the non-complexed amino groups, it isdesirable to use a divalent transition metal salt. When monovalenttransition metal salts are used, e.g. cuprous acetate, the cuprousacetate complex of available neighboring hydroxyl and amino group pairsis formed in poor yields and is weakly bound so that when theN-acylation step is carried out, N-acylation occurs at the sites ofweakly complexed amino groups (albeit at a somewhat slower rate) as wellas at the non-complexed amino group sites resulting in mixtures ofproducts and poorer yields of desired product.

The process of this invention is carried out in an inert organic solventwhich includes any organic solvent in which the polyamino-organiccompound and transition metal salt as well as the resultingpolyamino-organic compound-transition metal salt complex intermediateare reasonably soluble, and which will not react to any great extentwith the reagents of this process. Preferred solvents are polar, aproticorganic solvents, particularly dialkylamides (e.g. dimethylformamide)and dialkylsulfoxides (e.g. dimethylsulfoxide). Polar protic solventssuch as lower alkanols, particularly methanol and ethanol, may also beused in our process when such solvents are required for solubilityreasons. When using protic solvents in our process, it is usuallydesirable to employ a greater quantity of divalent transition metal saltthan when using aprotic solvents since protic solvents weaken thecomplexed functions in the polyamino-organic compound-transition metalsalt complex.

It is preferable to utilize substantially anhydrous solvents in ourprocess in order to obtain maximum yields and maximum complexingstability of the polyamino-organic compound-transition metal saltcomplex intermediates and, thus, to obtain maximum yield of desiredselectively blocked N-acyl derivatives of the non-complexed amines inthe polyamino compound. However, water may be present (and is frequentlydesirable for solubility reasons) even in amounts up to about 25% (byvolume) and higher without affecting the yields of selectively blockedN-acylated polyamino product, provided additional divalent transitionmetal salt is employed since water, being a protic solvent, also weakensthe complexed functions in the polyamino-organic compound transitionmetal salt complex intermediates. When too much water is employed, thepolyamino-organic compound-transition metal salt complexes are destroyedto a greater extent so that, when the final N-acylation step is carriedout, the amines having available neighboring hydroxyl groups are notsufficiently protected by their transition metal salt complexes and willalso react with the acylating reagent as well as the non-complexed aminogroups resulting in a mixture of products similar to those obtained whenthe acylating reaction is carried out in the absence of thepolyamino-organic compound-transition metal salt complexes.

Thus, in our process, an equilibrium appears to exist between thepolyamino-organic compound having at least one available neighboringamino/hydroxy group pair [A], tthe transition metal salt (e.g. cupricacetate, Cu(OAc)₂) and the solvent which may be indicated as follows:##EQU1##

With an aprotic solvent, the equilibrium favors the formation of atransition metal salt complex of a polyamino-organic compound havingavailable, neighboring amino/hydroxyl groups, while an organic proticsolvent or the presence of excess water in an organic aprotic solventfavors decomplexing of the polyamino-organic compound transition metalsalt complex. Thus, in the latter case, in order to obtain high yieldsof the polyamino-organic compound-salt complex in solution, greaterquantities of transition metal salt are required to force theequilibrium in the desired direction than is necessary when anessentially anhydrous aprotic solvent is used.

Usually, when carrying out the process of this invention, the molarquantity of transition metal salt used in the first step of the processis at least equal to the molar quantity of the polyamino-organiccompound times the number of available, neighboring amino- and hydroxylgroup pairs therein, and the molar quantity of acylating agent used inthe second step of our process is about equal to the molar quantity ofthe polyamino-organic compound times the number of non-complexed aminofunctions in the molecule which are to be protected. Whether or notaprotic or protic organic solvents are employed or water is present inthe reaction mixture, the molar quantity of N-acylating reagent in thesecond step of our in situ process always remains about equal to themolar quantity of polyamino-organic compound times the number ofnon-complexed amino groups which are to be N-acylated.

While the molar quantity of N-acylating reagent is usually equal to themolar quantity of the polyamino-organic compound multiplied by thenumber of all the non-complexed amines in the molecule, when thereexists a difference in the reactivity of the non-complexed amines, onemay use less N-acylating reagent if one desires to N-acylate only themore reactive amines. Thus, for example, upon reaction of sisomicin withat least two moles of a transition metal salt (e.g. cupric acetate) inan inert solvent (e.g. dimethylsulfoxide), there is obtained asisomicin-transition metal salt complex (e.g. sisomicin-cupric acetatecomplex) wherein the transition metal salt forms a complex with the3"-amino function-4"-hydroxyl group pair and with the1-amino-2"-hydroxyl group pair leaving three amino groups non-complexed,i.e. those at the 3, 2' and 6' positions. Reaction of the foregoing insitu with about three mole of N-acylating reagent (e.g. aceticanhydride) followed by removal of the transition metal cation (e.g.copper II) by reaction with hydrogen sulfide yields a tri-N-acylderivative, e.g. 3,2',6'-tri-N-acetylsisomicin. Alternatively, reactionof the corresponding sisomicin-nickel (II) acetate complex in methanolwith only two moles of N-acylating reagent (e.g.N-(2,2,2-trichloroethoxycarbonyloxy)succinimide) yields the2',6'-di-N-acyl derivative (e.g.2',6'-di-N-(2,2,2-trichloroethoxycarbonylsisomicin) in excellent yields,the more reactive 2' and 6' amino groups having been preferentiallyN-acylated over the less reactive 3-amino group under these conditions.

In our process, after the polyamino-organic compound-transition metalsalt complex has been reacted with an acylating agent to N-acylate thenon-complexed amino groups, the transition metal cation is thenconveniently removed from the N-acylated polyamino-organiccompound-transition metal salt complex by means of a transition metalprecipitating reagent or by means of ammonium hydroxide. In the lattercase, the transition metal cation is removed by the preferentialformation of a complex with ammonium hydroxide which is soluble inammonium hydroxide and water. This method of removing the transitionmetal cation is convenient when the selectively blocked N-acylpolyamino-organic derivative is soluble in organic solvents since it maybe then extracted from the aqueous organic solvent mixture whichcontains the ammonium hydroxide transition metal salt complex.

In carrying out our process, it is usually more convenient to remove thetransition metal cation by means of precipitating reagents known in theart. Useful precipitating reagents include dioxime of dimethylglyoxal,1,3-dicarbonyl-alkanes such as acetylacetone and heptane-3,5-dione aswell as sulfide precipitating reagents such as ammonium sulfide, alkalimetal sulfides (e.g. sodium sulfide), alkaline earth metal sulfides(e.g. calcium sulfide), alkaline earth metal hydrosulfides (e.g. sodiumhydrosulfide) and hydrogen sulfide. Of the foregoing, particularlyuseful precipitating reagents are dioxime of dimethylglyoxal,acetylacetone and hydrogen sulfide.

When removing the transition metal cation, hydrogen sulfide is aprecipitating reagent of choice since it is a simple procedure to merelybubble hydrogen sulfide through the reaction mixture; also, theresulting transition metal sulfide is completely precipitated in a shortperiod of time and is easily removed by filtration.

The acyl blocking groups, Y, and the corresponding acylating reagentswhereby they are formed are well known in the art as well as methods fortheir removal after a desired chemical transformation has been carriedout at some other site in the molecule. In this application and in theclaims, acyl blocking groups, Y, which may be selectively introduced insitu onto non-complexed amino functions in a polyamino-organic compoundtransition metal salt complex intermediate are contemplated as includingbenzyloxycarbonyl and substituted benzyloxycarbonyl groups such asp-nitrobenzyloxycarbonyl and p-methoxybenzyloxy-carbonyl (which areeasily removable by catalytic reduction); aryloxycarbonyl groups such asphenoxycarbonyl; and alkoxycarbonyl groups such as methoxycarbonyl,ethoxycarbonyl and the like (which are preferentially removed by basichydrolysis); trichloroethoxycarbonyl groups (removable by zinc in aceticacid); tertiaryalkoxycarbonyl groups such as tert.-butoxycarbonyl andtert.-amyloxycarbonyl groups (removable by mild acid hydrolysis);halogenalkylcarbonyl groups such as chloroacetyl (removable with base orwith thiourea or a similar reagent) and trifluoroacetyl (easily removedunder mild basic conditions); succinimido and phthalimido groups (easilyremoved by means of hydrazine) and alkanoyl groups such as acetyl,propionyl, and the like as well as aroyl groups such as benzoyl (whichare removed by basic hydrolysis). The reaction conditions necessary tointroduce any of the foregoing N-acyl derivatives on a free aminofunction are well documented in the literature and within the knowledgeand expertise of one skilled in the art of amine chemistry.

Polyamino-organic compounds useful as starting compounds in our processinclude any polyamino-organic compound which has at least one aminofunction which has an available neighboring hydroxyl group and whichalso has at least one amino function which is either devoid of anavailable neighboring hydroxyl group and therefore cannot form a complexwith a transition metal salt or which is sterically less available to aneighboring hydroxyl so that any salt complex formed at that site wouldbe less stable than other strongly complexed amino/hydroxyl pairs, thusrendering the amino function available to N-acylation in the second stepof our process.

The polyamino-organic starting compounds of our process may besubstituted by groups other than hydroxyl groups and may contain heretoatoms provided they do not react with transition metal salts or with theacylating reagents. Thus, the polyamino-organic starting compounds ofthis process are preferably devoid of sulfhydryl and mercaptyl groups.

Contemplated as included among the polyamino-organic starting compoundsof our process are monocyclic-, bicyclic- and tricyclic-polyaminoarylhydroxides such as ##STR1## as well as polyaminocycloalkanols such as(cis,cis,cis)-2,4-diaminocyclohexanol and(trans,cis,trans)-2,4-diaminocyclo-hexanol and acyclic polyaminocompounds such as 2,6-diamino-hexanol.

In acyclic polyamino-organic starting compounds of our process, aminoand hydroxyl groups are neighboring and available to each other whichare positioned so that the carbon chain (or hetero carbon chain)containing the amino/hydroxyl group pairs can form a five, six, or sevenmembered ring together with the transition metal cation.

Our process is particularly useful in the carbohydrate art when carryingout chemical transformations of pseudodisaccharides such as4-O-(6'-amino-6'-deoxyglucosyl)deoxystreptamine, neamine, garamine,kanamine, paromamine, and the gentamines (e.g. C₁, C_(1a), C₂, C_(2b),etc.).

Our process finds its greatest use, however, in theaminocyclitol-aminoglycoside art where chemical transformations ofaminoglycoside antibiotics are continually being made to synthesize newderivatives having enhanced antibacterial activity and/or a morefavorable antibacterial spectrum. Most of these transformations aremultistep, low yield processes in which several initial steps aredirected merely to protecting amino and/or hydroxyl functions withblocking groups prior to carrying out the main chemical transformationstep, in an attempt to improve the yield of desired transformationproduct. By our process, the amino groups which require protection areselectively blocked in excellent yield in a simple two-step-one-vesselprocedure.

4,5-Linked aminoglycoside antibiotics containing deoxystreptamine whichare useful starting compounds of our process include ribostamycin (inwhich the 1-amino-6-hydroxyl group pair is converted in situ to atransition metal salt complex, thence all other amino functions areN-acylated followed by removal of the transition metal salt, thencemethylation of the resulting 1-N-unsubstituted-poly-N-acylribostamycinutilizing known methylation techniques to produce 1-N-methylribostamycinhaving antibacterial activity); xylostamycin, 3'-deoxyribostamycin, and3',4',5"-trideoxyribostamycin. Other useful starting compounds include4-O-linked-streptamine containing aminocyclitol-amino-glycosides such as1,3-di-de-N-amidinodihydrostreptomycin which, when reacted with at leasttwo equivalents of cupric acetate, forms in situ a cupric acetatecomplex involving the 1-amino and 6-hydroxy group pair and the 3-aminoand 2-hydroxyl group pair so that, upon reaction with an acylatingreagent followed by removal of the cupric cation, a2"-N-acyl-1,3-di-de-N-amidino-dihydrostreptomycin derivative is formedexclusively and in excellent yields. On the other hand, if a transitionmetal salt is not added to the 1,3-di-de-N-amidinodihydrostreptomycinprior to treatment with an acylating reagent, there is formed a mixtureof products of which the 1-N-acyl derivative is predominant.

The preferred mode of our process is that wherein amino groups areselectively blocked in a 4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitolantibacterial agent. By the novel process of this invention, it is nowpossible to easily prepare in good yields selectively blockedaminoglycoside antibiotic derivatives such as 2',6'-di-N-acyl,3,6'-di-N-acyl, 3,2',6'-tri-N-acyl, and 1,3,2',6'-tetra-N-acylderivatives of 4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol derivativesby a two-step-one-vessel process, all of which were heretofore unknownand which are valuable as intermediates in preparing antibacteriallyactive transformation products of the parent aminoglycoside.

A particularly valuable mode of our process is that whereby is prepareda 3,2',6'-tri-N-acyl derivative of a4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol such as sisomicin,tobramycin, verdamicin, gentamicin C₁, gentamicin C_(1a) and gentamicinC₂, which are valuable intermediates in preparing the corresponding1-N-alkylaminoglycosides (after removal of the N-acyl groups at the 3,2' and 6' positions) which are valuable antibacterial agents describedin Belgium Pat. No. 818,431, South African Pat. No. 74/4939, and U.S.Pat. No. 4,002,742. Of the foregoing, 3,2',6'-tri-N-acylsisomicins,particularly 3,2',6'-tri-N-acetylsisomicin, are of great value asintermediates in preparing 1-N-ethylsisomicin, a potent anti-bacterialagent having an improved antibacterial spectrum over that of itsprecursor antibiotic, sisomicin.

In a preferred mode of preparing a3,2',6'-tri-N-acyl-aminocyclitol-aminoglycoside (e.g.3,2',6'-tri-N-acetylsisomicin) by our process, sisomicin is dissolved inan inert solvent (e.g. aqueous dimethylformamide) to which is added adivalent transition metal salt (e.g. cupric acetate hydrate) in anamount equal to about 15 molar equivalents per mole of sisomicin (twomolar equivalents being required to form transition metal salt complexeswith the 1-amino-2"-hydroxyl group pairs and with the3"-amino-4"-hydroxyl group pairs and additional excess transition metalsalt being required because of the presence of water) and the reactionstirred at room temperature (usually about 30 minutes), thus completingthe first step of our two-step in situ process. To the thus preparedsolution of aminocyclitol-aminoglycoside-divalent-transition metal saltcomplex (e.g. sisomicin-cupric acetate complex) is added an acylatingreagent (e.g. acetic anhydride) in an amount equal to about three molarequivalents per mole of aminoglycoside derivative (e.g. sisomicin-cupricacetate salt complex) (there being three non-complexed amino groups atpositions 3, 2' and 6') and the reaction mixture is stirred at roomtemperature until the reaction is complete (usually in about 30 minutes)as evidenced by a thin layer chromatogram. The transition metal cation(e.g. copper II) is then easily removed by bubbling hydrogen sulfidethrough the reaction mixture followed by separation of the resultingprecipitate of the sulfide salt of the transition metal (e.g. cupricsulfide) by filtration to obtain a filtrate comprising the3,2',6'-tri-N-acyl-aminocyclitol-aminoglycoside (e.g.3,2',6'-tri-N-acetyl-sisomicin) which may be used without isolation orpurification as intermediates to prepare the corresponding1-N-substituted derivatives, specifically the corresponding 1-N-alkylderivatives utilizing procedures such as described in South African Pat.No. 74/4939 and in copending U.S. Ser. No. 492,998 filed July 30, 1974of common assignee as the instant application, now U.S. Pat. No.4,002,742, the subject matter of which is incorporated herein byreference. Alternatively, the 3,2',6'-tri-N-acyl derivative may bepurified and isolated utilizing conventional techniques such asevaporating the solvent and chromatographing a solution of the resultantresidue on a column of silica gel.

In carrying out our process, we have found that mixtures of transitionmetal salt complexes are sometimes advantageously employed to producegood yields of selectively N-acylated polyamino compounds. It isbelieved this is due to the fact that some transition metal salts formmore strongly bound transition metal salt complexes with certainavailable, neighboring amino/hydroxyl group pairs than do othertransition metal salts. Thus, when preparing3,2',6'-tri-N-acetylsisomicin, improved yields are obtained when theaforedescribed process is carried out in an aprotic solvent (e.g.dimethylsulfoxide) utilizing as divalent transition metal salt anequimolar mixture of nickel (II) acetate and cupric acetate (the totalmolar equivalents of transition metal salts being four times that ofsisomicin) followed by reaction in situ of the sisomicin-nickel (II)acetate-cupric acetate complex thereby formed with about three molarequivalents of acetic anhydride per mole of sisomicin, thence removal ofthe cupric and nickel (II) ions by precipitation as the sulfide saltsand thence isolation and purification of the resulting3,2',6'-tri-N-acetylsisomicin in yields of at least 90% theory ascompared with a 76% theoretical yield when cupric acetate was usedalone.

A particularly valuable mode of our process is that utilizing cobalt(II) acetate as transition metal whereby is obtained almost theoreticalyields of essentially pure selectively blocked N-acylated derivatives.For example, treatment of sisomicin with about two equivalents of cobalt(II) acetate in an aprotic solvent such as dimethylsulfoxide ordimethylformamide followed by treatment in situ of the resultingsisomicin.di-cobalt (II) acetate complex thereby formed with about threeequivalents of acetic anhydride followed by removal of the cobalt ionfrom the tri-N-acetylsisomicin.di-cobalt (II) acetate complex bytreatment with hydrogen sulfide, thence isolation of the selectivelyblocked sisomicin thereby formed yields 3,2',6'-tri-N-acetylsisomicin ofhigh purity in near quantitative yields.

In contrast to the foregoing, prior art acylation procedures utilizingthree moles of acetic anhydride per mole of sisomicin without the use ofcupric (II), nickel (II) or cobalt (II) ion complexes, produce a mixtureof products containing penta-N-acetylsisomicin, mixtures oftetra-N-acetylsisomicins, mixtures of tri-N-acetylsisomicins along withother products from which the desired 3,2',6'-tri-N-acetylsisomicincould not be isolated free from co-produced products.

In the above procedure, by modifying the quantity of transition metalsalt and acylating reagent and (incidentally) changing the solvent, onecan obtain good yields of 1,3,2',6'-tetra-N-acetylsisomicin instead of3,2',6'-tri-N-acetylsisomicin. Thus, by adding from about a half to anequimolar equivalent amount of divalent transition metal salt (e.g.cupric acetate hydrate) per mole of sisomicin in an aqueous methanolicsolution, there is obtained in situ a sisomicin-cupric acetate complexinvolving the 3"-amino-4"-hydroxyl group pair. Reaction of the foregoingin situ with about 4 molar equivalents of acetic anhydride followed byremoval of the cupric cation by precipitation as the sulfide salt andthence isolation and purification utilizing known techniques yields1,3,2',6'-tetra-N-acetylsisomicin in yields of almost 80% theory, anintermediate useful when transformation at the 3"-amino group isdesired. Without the presence of cupric (II), nickel (II) or cobalt (II)ion in the reaction mixture, the foregoing reaction will produce muchlower yields of the product.

In our process, after preparation of the polyamino-organiccompound-transition metal salt complex, one can prepare stepwise in situmixed N-acyl derivatives of the remaining non-complexed amino groupswhen said amines have different degrees of reactivity without having toisolate the first N-acyl derivative prior to preparation of the desiredmixed N-acyl derivative. Depending upon the types of N-acyl groupsintroduced into the molecule and their relative ease of removal, it ispossible, by our process, to prepare any desired combination ofselectively N-acylated polyamino-organic compound. Thus, for example, bymodifying the procedure described hereinabove for the preparation of3,2',6'-tri-N-acetylsisomicin, it is possible to prepare otherselectively N-acylated derivatives thereof such as1,2',3-tri-N-acetylsisomicin or 2',3-di-N-acetylsisomicin.

Specifically, upon reaction of sisomicin in 95% aqueous methanol withabout an equimolar amount of cupric acetate, there is obtained in situ asisomicin-cupric acetate complex essentially involving the 3"-amino and4"-hydroxyl groups. Reaction of the foregoing sisomicin-cupric acetatecomplex with about a molar equivalent of ethylthioltrifluoro acetateproduces a monoacyl derivative of the most reactive non-complexed aminofunction therein; namely, the 6'-amino, to produce in situ6'-N-trifluoroacetylsisomicin-cupric acetate complex. By subsequentlyadding about three molar equivalents of acetic anhydride to theforegoing derivative in situ, the remaining three non-complexed aminogroups are N-acetylated to produce1,3,2'-tri-N-acetyl-6'-N-trifluoroacetylsisomicin-cupric acetatecomplex. After destroying the cupric acetate complex by bubblinghydrogen sulfide through the reaction mixture and separating theresulting cupric sulfide by filtration, evaporation of the filtratefollowed by treatment of the residue containing1,3,2'-tri-N-acetyl-6'-N-trifluoroacetylsisomicin with ammoniumhydroxide causes hydrolysis of the 6'-N-trifluoroacetyl group withoutdisturbing the N-acetyl groups. Isolation of the resulting1,3,2'-tri-N-acetylsisomicin is easily effected by known techniques suchas evaporating the solution and chromatographing the resultant residue.The 1,3,2'-tri-N-acylaminoglycosides are valuable intermediates inpreparing 6'-N-alkylaminoglycosides, useful antibacterial agents such asthose described in co-pending applications of common assignee, Ser. No.596,799 filed July 17, 1975, and Ser. No. 666,715 filed Mar. 15, 1976.

Additionally, by forming in situ 6'-N-trifluoroacetylsisomicin-cupricacetate nickel (II) acetate complex in dimethylsulfoxide and adding twoequivalents of acetic anhydride, there is formed a di-N-acetylderivative involving the two more reactive non-complexed amino functionsat the 3- and 2'-positions to produce in situ3,2'-di-N-acetyl-6'-N-trifluoroacetylsisomicin-cupric acetate complex,which, after removal of the complex with hydrogen sulfide and hydrolysisof the 6'-N-trifluoroacetyl group by means of mild basic hydrolysis,yields 3,2'-di-N-acetylsisomicin. The3,2'-di-N-acetylaminocyclitol-aminoglycosides such as3,2'-di-N-acetylsisomicin are valuable intermediates in the preparationof 1,6'-di-N-alkylaminoglycosides, e.g. 1,6'-di-N-ethylsisomicin,valuable antibacterial agents described in co-pending application ofcommon assignee, Ser. No. 628,637 filed Nov. 4, 1975.

In yet another mode of our process, mixed N-acyl derivatives of apolyamino-organic compound may be prepared by introducing, in situ, someN-acyl derivatives on the polyamino-organic compound-transition metalsalt complex, then destroying the complex (such as with hydrogen sulfideor ammonium hydroxide) followed by N-acylation of the amino functionswhich formerly had been rendered inactive due to the presence of thecomplexes. By utilizing this procedure, it is possible to obtain a1,3,3"-tri-N-acetylsisomicin derivative, a useful intermediate for thepreparation of 2',6'-di-N-substituted derivatives such as2',6'-di-N-alkylsisomicin having antibacterial activity. Thus, forexample, treatment of sisomicin with about 2 to 3 molar equivalents ofnickel (II) acetate in methanol yields in situ a sisomicin-nickel (II)acetate complex involving the 3"-amino-4"-hydroxyl group pair and the1-amino-2"-hydroxy group pair. Reaction of the sisomicin-nickel (II)acetate complex in situ with about two moles ofN-tert.-butoxycarbonyloxyphthalimide produces a di-N-acyl derivativeinvolving the more reactive non-complexed 2' and 6'-amino groups ascompared with the non-complexed 3-amino group to produce2',6'-di-N-tert.-butoxycarbonylsisomicin-nickel (II) acetate complex.Removal of the nickel (II) ion as nickel sulfide according to ourprocess followed by reaction in situ of the resulting filtratecontaining 2',6'-di-N-tert.-butoxycarbonylsisomicin with aceticanhydride yields1,3,3"-tri-N-acetyl-2',6'-di-N-tert.-butoxycarbonylsisomicin which, uponcontrolled acid hydrolysis in situ such as by 5% trifluoroacetic acid intetrahydrofuran yields 1,3,3"-tri-N-acetylsisomicin.

Alternatively, the 2',6'-di-N-tert.-butoxycarbonylsisomicin intermediateprepared by the above procedure may be isolated per se, then reducedwith lithium aluminum hydride in tetrahydrofuran to produce2',6'-di-N-methylsisomicin (i.e. 2'-N-methyl-Antibiotic G-52) useful asantibacterial agent.

The above discussion describing the preparation of selectivelyN-acylated sisomicin derivatives, particularly valuable intermediatesprepared by our novel process, illustrates the flexibility andversatility of our process. Other particularly valuable intermediatesprepared by our process are the 6'-N-acyl and 3,6'-di-N-acyl derivativesof aminocyclitol-amino glycosides such as gentamicin B, gentamicin B₁and of kanamycin A, which are also useful intermediates in thepreparation of 1-N-substituted derivatives, particularly the1-N-(γ-amino-α-hydroxybutyryl)- and the1-N-(β-amino-α-hydroxypropionyl)- and the1-N-(δ-amino-α-hydroxyvaleryl)- derivatives of gentamicins B and B₁ andof kanamycin A, all of which are valuable antibacterial agents.

It is to be noted that the foregoing1-N-(aminohydroxyalkanoyl)aminocyclitol-aminoglycosides may be in theR,S- form or in the R- form or in the S-form. In accordance with thisinvention, each of the foregoing names includes all three forms. Thus,for example, the name 1-N-(γ-amino-α-hydroxybutyryl)gentamicin Bincludes 1-N-(S-γ-amino-α-hydroxybutyryl)gentamicin B,1-N-(R-γ-amino-α-hydroxybutyryl)gentamicin B and1-N-(R,S-γ-amino-α-hydroxybutyryl)gentamicin B and the name1-N-(β-amino-α-hydroxypropionyl)gentamicin B includes1-N-(S-β-amino-α-hydroxypropionyl)gentamicin B,1-N-(R-β-amino-α-hydroxypropionyl)gentamicin B, and1-N-(R,S-β-amino-α-hydroxypropionyl)gentamicin B.

By means of the novel 3,6'-di-N-acyl derivatives of gentamicins B andB₁, and of kanamycin A, prepared via the transition metal complexes bythe process of this invention, each of gentamicins B and B₁ andkanamycin A can now be converted to the corresponding1-N-aminohydroxyalkanoyl derivatives in high yields of pure product.Thus, for example, kanamycin A is convertible to3,6'-di-N-benzyloxycarbonylkanamycin A via the di-nickel (II) acetatecomplex in dimethylsulfoxide in over 80% theoretical yield. Reaction ofthe foregoing 3,6'-di-N-acyl derivative withN-(S-γ-benzyloxycarbonylamino-α-hydroxybutyryloxy)succinimide followedby treatment of the resulting1-N-(S-γ-benzyloxycarbonylamino-α-hydroxybutyryl)-3,6'-di-N-benzyloxycarbonylkanamycinA with hyrogen and a catalyst to remove the benzyloxycarbonyl protectinggroups produces 1-N-(S-γ-amino-α-hydroxybutyryl)kanamycin A (also knownas amikacin and as Antibiotic BB-K-8) of high purity in excellentoverall yields of over 50%.

The foregoing process of this invention thus represents a greatimprovement over the prior art methods of converting kanamycin A toamikacin such as that disclosed in J. Antibiotics 25, 695-708 (1972)whereby kanamycin A is first converted to the 6'-N-benzyloxycarbonylderivative in 45% yields which, when taken through essentially the sameconversions described hereinabove produces purified amikacin in overallyields of less than 10%. Similarly, by the method of our invention,kanamycin A via the 3,6'-di-N-benzyloxycarbonyl intermediate isconvertible to 1-N-(β-amino-α-hydroxypropionyl)kanamycin A (also knownas 1-N-isoserylkanamycin A) in overall yields greater than 50% theory ascompared with overall yields of less than 8% via prior art methodsutilizing the 6'-N-tert.-butoxycarbonyl intermediate such as describedin U.S. Pat. No. 3,939,143.

Similarly, by reacting gentamicin B in dimethylsulfoxide with about 3molar equivalents of cupric acetate there is obtained in situ thecorresponding gentamicin B-cupric acetate complex involving the3"-amino-4"-hydroxyl and the 1-amino-2"-hydroxyl group pairs. Reactionof the foregoing in situ with about 1 molar equivalent ofN-tert.-butoxycarbonyloxyphthalimide yields the corresponding6'-N-tert.-butoxycarbonylgentamicin B-di-cupric acetate complex, whilereaction with at least two moles of theN-tert.-butoxycarbonyloxyphthalimide yields the corresponding3,6'-di-N-tert.-butoxycarbonylgentamicin B-di-cupric acetate complex.Reaction of each of the foregoing with hydrogen sulfide followed byseparation of the resulting cupric sulfide by filtration yields asolution comprising 6'-N-tert.-butoxycarbonylgentamicin B or3,6'-di-N-tert.-butoxycarbonylgentamicin B, respectively. Each of theforegoing may be isolated from their respective reaction solutions andpurified in known manner and thence reacted withN-(S-γ-benzyloxycarbonylamino-α-hydroxybutyryloxy)succinimide or withN-(S-β-benzyloxycarbonylamino-α-hydroxypropionyloxy)succinimide or withN-(S-δ-amino-α-hydroxyvaleryl)succinimide according to procedures suchas described in the Examples and in the prior art to obtain1-N-(S-γ-amino-α-hydroxybutyryl)gentamicin B, or1-N-(S-β-amino-α-hydroxypropionyl)gentamicin B, or1-N-(S-δ-amino-α-hydroxyvaleryl)gentamicin B, respectively, all of whichare valuable antibacterial agents.

Alternatively, one may carry out the chemical conversion at C-1 in situwithout isolating the 3,6'-di-N-tert.-butoxycarbonylgentamicin B toobtain the corresponding 1-N-(S-γ-amino-α-hydroxybutyryl)gentamicin B or1-N-(S-β-amino-α-hydroxypropionyl)gentamicin B or1-N-(S-δ-amino-α-hydroxyvaleryl)gentamicin B in excellent overall yieldsof about 50% theory. The foregoing also represents a significantimprovement over prior art methods for preparing these compounds such asdescribed by P. J. L. Daniels et al in J. Antibiotics 27, 889 (1974)which produce said 1-N-aminohydroxyalkanoylgentamicin C₁ derivatives inless than 15% yields.

It is to be noted, also, that by our process the yield of6'-N-tert.-butoxycarbonylgentamicin B is nearly quantitative andrequires no chromatographic purification in contrast to prior artprocedures which give only about 46% yields and requires chromatographicisolation.

By our invention we have also discovered that, when using our novel3,6'-di-N-acyl-gentamicin B intermediates, e.g.3,6'-di-N-benzyloxycarbonylgentamicin B, in the conversion of gentamicinB to a 1-N-(aminohydroxyalkanoyl) derivative thereof, e.g.1-N-(β-amino-α-hydroxypropionyl)gentamicin B, reaction of the3,6'-di-N-acyl derivative with a racemic reagent, e.g.N-(β-benzyloxycarbonylamino-α-hydroxypropionyloxy)succinimide will yielda 3,6'-di-N-acyl-1-N-(R,S-aminohydroxyalkanoyl)gentamicin Bdiastereoisomeric mixture (e.g.3,6'-di-N-(benzyloxycarbonyl)-1-N-(R,S-β-benzyloxycarbonylamino-.alpha.-hydroxypropionyl)gentamicinB) which, surprisingly, is easily separated via chromatographictechniques into each diastereoisomer, e.g. into3,6'-di-N-benzyloxycarbonyl-1-N-(R-β-benzyloxycarbonylamino-α-hydroxypropionyl)gentamicinB and3,6'-di-N-(benzyloxycarbonyl)-1-N-(S-β-benzyloxycarbonylamino-α-hydroxypropionyl)gentamicinB which, upon removal of the 3,6'-di-N- blocking groups yields therespective diastereoisomeric antibacterial agent, e.g.1-N-(R-β-amino-α-hydroxypropionyl)gentamicin B and1-N-(S-β-amino-α-hydroxypropionyl)gentamicin B, respectively.Alternatively, removal of the protecting groups from3,6'-di-N-(benzyloxycarbonyl)-1-N-(R,S-β-benzyloxycarbonylamino-.alpha.-hydroxypropionyl)gentamicinB yields 1-N-(R,S,-β-amino-α-hydroxypropionyl)gentamicin B which can beobtained pure from impurities by chromatography on silica gel but whichis not thereby separated into the respective diastereoisomers. Thus, bymeans of our novel 3,6'-di-N-acyl intermediates, it is possible toproduce both diastereoisomeric derivatives of a1-N-(aminohydroxyalkanoyl)gentamicin B using a racemic reagent whereasheretofore, in order to obtain a given diastereoisomeric derivative, itwas necessary to utilize the corresponding reagent enantiomer.

Described above are some preferred modes of carrying out the process ofour invention, specific illustrations of which are more fully describedin the Examples. Equivalents thereof will become readily apparent to oneskilled in the art from the instant disclosure, said equivalents beingconsidered as part of Applicants' invention which is not to be construedas limited to the specific illustrations described herein and in theExamples.

DESCRIPTION OF THE COMPOSITION-OF-MATTER ASPECTS OF THE INVENTION

Included within the composition-of-matter aspects of this invention arenovel selectively blocked aminocyclitol-aminoglycoside derivatives whichare prepared by our process involving the in situ complexing ofneighboring available amino/hydroxyl group pairs with transition metalsalts. As discussed hereinabove, the selectively blocked N-acylderivatives of this invention are valuable intermediates in thepreparation of anti-bacterially active derivatives of saidaminocyclitol-aminoglycosides.

One of the composition-of-matter aspects of this invention resides inthe concept of

A 3,2',6'-tri-N-acyl-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitolselected from the group consisting of

3,2',6'-tri-N-Y-sisomicin, 3,2',6'-tri-N-Y-verdamicin,

3,2',6'-tri-N-Y-tobramycin, 3,2',6'-tri-N-Y-gentamicin C₁,

3,2',6'-tri-N-Y-gentamicin C_(1a), 3,2',6'-tri-N-Y-gentamicin C₂,

3,2',6'-tri-N-Y-gentamicin C_(2a), 3,2',6'-tri-N-Y-gentamicin C_(2b),

3,2',6'-tri-N-Y-Antibiotic 66-40B,

3,2',6'-tri-N-Y-Antibiotic 66-40D,

3,2',6'-tri-N-Y-Antibiotic JI-20A,

3,2',6'-tri-N-Y-Antibiotic JI-20B,

3,2',6'-tri-N-Y-Antibiotic G-52,

the 5-epi- and 5-epi-azido-5-deoxy analogs of the foregoing;

3,2',6'-tri-N-Y-kanamycin B,

3,2',6'-tri-N-Y-3',4'-dideoxykanamycin B,

3,2',6'-tri-N-Y-nebramycin factor 4,

3,2',6'-tri-N-Y-nebramycin factor 5',

3,2',6'-tri-N-Y-3',4'-dideoxy-3',4'-dehydrokanamycin B,

3,2',6'-tri-N-Y-3',4'-dideoxy-6'-N-methylkanamycin B, an

3,2',6'-tri-N-Y-5-deoxysisomicin;

wherein Y is an acyl group, including acyl functions describedhereinabove.

The foregoing derivatives are particularly valuable as intermediates inthe preparation of the corresponding 1-N-alkyl derivatives thereofwhich, after removal of the 3,2',6'-tri-N-acyl blocking groups arepotent antibacterial agents as described in South African Pat. No.73/4939, and in U.S. application Ser. No. 492,998 filed July 30, 1974 ofcommon assignee now U.S. Pat. No. 4,002,742. In brief, as illustrated inExamples 16 and 17 by reacting an acid addition salt of the3,2',6'-tri-N-acyl-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitols of thisinvention with an aldehyde followed by reduction in situ of theresulting Schiff-base derivative at C-1 by procedures analogous to thosedescribed in U.S. Pat. No. 492,998 filed July 30, 1974, now U.S. Pat.No. 4,002,742 there is obtained the corresponding1-N-alkyl-3,2',6'-tri-N-acyl-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitolwhich, after removal of the N-acyl derivatives results in much greateroverall yields (e.g. usually above 50% theory) of purer1-N-alkylaminoglycosides than obtained by presently known methods ofpreparing 1-N-alkylaminoglycosides (usually less than 20% theory).

For example, when preparing 1-N-ethylsisomicin by first convertingsisomicin to 3,2',6'-tri-N-acetylsisomicin via the transition metal saltcomplex in situ process of this invention followed by reaction of asulfuric acid addition salt of the resulting3,2',6'-tri-N-acetylsisomicin with acetaldehyde followed by reductionwith sodium cyanoborohydride, thence removal of 3,2',6'-tri-N-acetylfunctions via basic hydrolysis, there is obtained 1-N-ethylsisomicin ofhigh purity in yields of about 60% theory; whereas conversion ofsisomicin to 1-N-ethylsisomicin by the same process but withoutacetylating at the 3, 2' and 6' positions produces 1-N-ethylsisomicin inyields of about 11% theory. Thus, of particular value in preparing1-N-alkylaminocyclitol-aminoglycoside derivatives are the3,2',6'-tri-N-acylaminoglycosides wherein the "acyl" is lower alkanoyl,preferably acetyl. Of these, a particularly preferred species is3,2',6'-tri-N-acetylsisomicin, valuable intermediate in the preparationof 1-N-ethylsisomicin.

Other valuable 3,2',6'-tri-N-acylaminocyclitol-aminoglycosides are thosewherein Y is 2,2,2-trichloroethoxycarbonyl, a particularly preferredspecies being 3,2',6'-tri-N-(2,2,2-trichloroethoxycarbonyl)sisomicin, avaluable intermediate in the preparation of1-N-acyl-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitols, particularly1-N-acyl derivatives of sisomicin, e.g. 1N-acetylsisomicin, valuable asantibacterial agents and as intermediates in preparing the corresponding1-N-alkyl derivatives, said 1-N-acyl derivatives being described inco-pending applications of common assignee as the instant application,i.e. U.S. Ser. No. 452,586 and Ser. No. 452,571, both filed Mar. 19,1974.

Another composition-of-matter aspect of this invention is the concept ofa 2',6'-di-N-Y-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol selectedfrom the group consisting of

2',6'-di-N-Y-sisomicin, 2',6'-di-N-Y-tobramycin,

2',6'-di-N-Y-gentamicin C_(1a), 2',6'-di-N-Y-Antibiotic 66-40B,

2',6'-di-N-Y-Antibiotic 66-40D,

2',6'-di-N-Y-Antibiotic JI-20A,

the 5-epi- and 5-epi-azido-5-deoxy analogs of the foregoing;

2',6'-di-N-Y-kanamycin B,

2',6'-di-N-Y-3',4'-dideoxykanamycin B,

2',6'-di-N-Y-3',4'-dideoxy-3',4'-dehydrokanamycin B, and

2',6'-di-N-Y-5-deoxysisomicin; wherein Y is an acyl group.

Particularly valuable derivatives of the foregoing are those wherein Yis 2,2,2-trichloroethoxycarbonyl or tert.-butoxycarbonyl, particularly2',6'-di-N-(2,2,2-trichloroethoxycarbonyl)sisomicin and2',6'-di-N-(tert.-butoxycarbonyl)sisomicin, valuable intermediates inpreparing 2',6'-di-N-alkyl-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitolshaving antibacterial activity, e.g. 2',6'-di-N-ethylsisomicin.

Still another composition-of-matter aspect of this invention resides inthe concept of a3,6'-di-N-acyl-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol selectedfrom the group consisting of

3,6'-di-N-Y-kanamycin A, 3,6'-di-N-Y-gentamicin B,

3,6'-di-N-Y-gentamicin B₁, 3,6'-di-N-Y-gentamicin A₃,

3,6'-di-N-Y-6'-N-methylkanamycin A wherein Y is an acyl group.

Particularly preferred compounds of this aspect of the invention arethose wherein Y is a member selected from the group consisting ofacetyl, tert.-butoxycarbonyl, benzyloxycarbonyl and2,2,2-trichloroethoxycarbonyl. When Y is acetyl, the foregoingderivatives are valuable mainly as intermediates in the preparation ofthe corresponding 1-N-alkyl derivatives via the procedure describedhereinabove and in Example 18B. When Y is such as tert.-butoxycarbonyl,benzyloxycarbonyl and 2,2,2-trichloroethoxycarbonyl, the foregoingcompounds are of particular value in the preparation of thecorresponding 1-N-(γ-amino-α-hydroxybutyryl),1-N-(β-amino-α-hydroxypropionyl) and 1-N-(δ-amino-α-hydroxyvaleryl)derivatives (as discussed in the Description of the Process Aspect ofthe Invention) which, when the 3,6'-di-N-acyl groups are removed, arepotent anti-bacterial agents known in the art.

Still another composition-of-matter aspect of our invention resides inthe concept of a polyamino-organic compound-transition metal saltcomplex wherein at least one of said amino groups has an availableneighboring hydroxyl group and said salt is a salt of a divalenttransition metal cation selected from the group consisting of copper(II), nickel (II), cobalt (II), and cadmium (II) or is a mixture of saidsalts, said polyamino-organic compound-transition metal salt complexhaving complex functions between said divalent transition metal salt andsaid available neighboring amino and hydroxyl group pairs, the number ofcomplex functions being no greater than the number of said availableneighboring amino and hydroxyl group pairs.

Usually, all the complex functions are the same in the polyamino-organiccompound-transition metal salt complexes of this invention; however, apolyamino-organic compound-transition metal salt complex may containdifferent complex functions, e.g. as in sisomicin-cupric acetate-nickel(II) acetate complex discussed hereinabove and specifically described inthe Examples.

The polyamino-organic compound-transition metal salt complexes areconveniently used as intermediates in situ in the solvent medium inwhich they are prepared, and therefore, there is usually no need toisolate the transition metal salt complexes of this invention. Thus,another aspect of our invention resides in the concept of a compositioncomprising a polyamino-organic compound-transition metal salt complex inan inert, organic solvent, particularlyaminocyclitol-aminoglycoside-transition metal salt complexes in an inertorganic solvent. The transition metal salt complexes can be isolated,however, either by evaporating the solvent medium in vacuo or byprecipitating the complex by pouring the solution thereof into ether,whereby is produced a polyamino-organic compound-transition metal saltcomplex as an amorphous powder which is characterized by colorsdifferent from the transition metal salt and of the polyamino-organiccompound precursor, and which are also characterized by infraredspectrum taken in the solid state. Furthermore, in solutions, thespectrum of the isolated transition metal salt complexes of thepolyamino-organic compound in the visible region is different from thatof either the transition metal salt alone or the polyamino-organiccompound alone.

Preferred transition metal salt complexes of this invention are thosederived from cupric acetate, nickel (II) acetate, cobalt (II) acetate ormixtures thereof, particularly those wherein the polyamino-organiccompound is an aminocyclitol-aminoglycoside, preferably a4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol such as those specificallydescribed in the Examples.

Of particular value are the transition metal salt complexes of4,6-di-O-(aminoglycosyl)-2-deoxystreptamines such as tobramycin,sisomicin, verdamicin, gentamicin C₁, gentamicin C₁ a, gentamicin B,gentamicin B₁, and kanamycin A, which contain available neighboringamino functions and hydroxyl group pairs at the 3" and 4"-positions andat the 1 and 2"-positions, respectively. When the transition metal saltcomplexes of the foregoing aminocyclitol-aminoglycosides are preparedwith at least 2 moles of transition metal salt per mole of4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol there will be complexfunctions involving the transition metal salt and both neighboring aminogroup/hydroxyl group pairs. Alternatively, reaction of any of theaforementioned 4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitols with butone molar equivalent of transition metal salt will produce apolyamino-organic compound-transition metal salt complex of thisinvention having but one complex function which is believed to be at thesite of the 3"-methylamino-4"-hydroxyl group pair. As discussed indetail hereinabove and as exemplified in the Examples, eachpolyamino-organic compound-transition metal salt complex is useful forpreparing different selectively blocked derivatives of the parentaminoglycoside. Thus, 4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitolshaving two complexing functions, e.g. sisomicin-dicupric acetatecomplex, upon reaction with three moles of acylating reagent accordingto our process will produce high yields of a3,2',6'-tri-N-acylsisomicin; whereas a4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol having but one complexingfunction therein (e.g. sisomicin-monocupric acetate complex) uponreaction with four moles of acylating reagent will produce almosttheoretical yields of 1,3,2',6'-tetra-N-acylsisomicin.

Included among the preferred polyamino-organic compound-transition metalsalt complexes of this invention are sisomicin-dicupric acetate complex,sisomicin-dicobalt (II) acetate complex, sisomicin-dinickel (II) acetatecomplex and the mixed sisomicin-cupric acetate-nickel (II) acetatecomplex, all of which are valuable intermediates in preparing3,2',6'-tri-N-acylsisomicin selectively blocked compounds which, inturn, are valuable intermediates in the preparation of1-N-alkylsisomicins (particularly 1-N-ethylsisomicin) and1-N-acylsisomicin (particularly 1-N-acetylsisomicin) valuableanti-bacterial agents.

Other particularly valuable polyamino-organic compound-transition metalsalt complexes of this invention are gentamicin B-dicupric acetatecomplex and kanamycin A-dinickel (II) acetate complex, valuableintermediates in preparing the 3,6'-di-N-acyl derivatives of gentamicinB and kanamycin A, e.g. 3,6'-di-N-tert.-butoxycarbonylgentamicin B and3,6'-di-N-benzyloxy-carbonylkanamycin A, in turn, valuable intermediatesin preparing the 1-N-(β-amino-α-hydroxypropionyl)-, the1-N-(γ-amino-α-hydroxybutyryl)-, and the 1-N-(δ-amino-α-hydroxyvaleryl)-derivatives of gentamicin B and of kanamycin A, valuable antibacterialagents.

DESCRIPTION OF THE PROCESS IMPROVEMENT ASPECT OF THE INVENTION

Another aspect of our invention resides in the concept of processimprovements which result when novel selectively blockedaminocyclitol-aminoglycosides of this invention are utilized asintermediates in processes known in the art. Particularly valuableprocess improvements include the improvement resulting from the use of3,2',6'-tri-N-acetylsisomicin as starting compound in the processdescribed in U.S. Ser. No. 492,998 filed July 30, 1974 whereby1-N-ethylsisomicin is prepared. Another particularly valuable processimprovement aspect of this invention is the improvement resulting fromthe use of 3,6'-di-N-acyl derivatives of kanamycin A and of gentamicinsB and B₁, in particular the 3,6'-di-N-tert.-butoxycarbonyl or the3,6'-di-N-benzyloxycarbonyl and the3,6'-di-N-(2,2,2-trichloroethoxycarbonyl) derivatives thereof asstarting compounds in known processes for preparing the1-N-(β-amino-α-hydroxypropionyl)-, the 1-N-(δ-amino-α-hydroxybutyryl)-,and the 1-N-(δ-amino-α-hydroxyvaleryl)- derivatives of kanamycin A andof gentamicins B and B₁.

As described in detail under the Description of theComposition-of-Matter Aspect of the Invention and in the Examples, byusing novel, selectively blocked N-acyl derivatives of our invention,greatly improved yields of purer 1-N-substituted aminoglycosides areobtained in fewer reaction steps than by known, prior art methods. Theseprocess improvements, resulting from the use of theaminocyclitol-aminoglycoside transition-metal salt complexes, are partof our overall inventive concept and are specifically defined in theclaims attached hereto.

The following Examples are illustrative of a preferred mode of carryingout our invention but are not to be construed as limiting the scopethereof. Equivalents thereof will be obvious to one skilled in the artreading this application and said equivalents are contemplated asincluded within this invention.

EXAMPLE 1 3,2',6'-TRI-N-ACETYLSISOMICIN A. Via Cupric Acetate Complex

Add cupric acetate hydrate (9 gms., 45 mmol) to a stirred solution ofsisomicin (1.3 gms., 2.9 mmol) in water (16 ml.) and dimethylformamide(54 ml.). Stir at room temperature for 35 minutes, then to the cupricsalt complex thereby formed add dropwise at a rate of about 25 drops perminute 9.3 ml. of a 1 molar solution of acetic anhydride indimethylformamide (9.3 mmol). Stir the reaction mixture for anadditional 30 minutes, then add 30 ml. of water and bubble hydrogensulfide through the solution for about 10 minutes, stir the mixture foran additional 30 minutes, then filter the solution through a pad ofCelite and wash the cupric sulfide residue with three 20 ml. portions ofwater. Concentrate the combined filtrate and water washings andchromatograph the resultant residue on silica gel (150 gms., 60-200mesh) eluting with chloroform: methanol:ammonium hydroxide (30:10:1).Combine like fractions as determined by thin layer chromatography onsilica gel using a solvent system consisting ofchloroform:methanol:ammonium hydroxide (2:1:0.34) and evaporate thefractions containing the major product in vacuo and lyophilize theresultant aqueous mixture to a residue comprising3,2',6'-tri-N-acetylsisomicin (1.29 gms., 76% yield); [α]_(D) ²⁶ +186.7° (c, 4.4 in water); pmr (ppm) (D₂ O): δ 1.22 (4"--C--CH₃); 1.94,1.98, 2.0 (N-Acetyls); 2.51 (3"--N--CH₃); 2.59 (H-3", J_(2"),3" = 9.5Hz); 5.10 (H-1", J_(1"),2" = 4.0 Hz); 5.51 (H-1', J_(1'),2' = 2.5 Hz);mass spectrum: (M⁺ °) m/e 573, also m/e 392, 374, 364, 346, 233, 215,205, 187; 160; 443, 425, 415, 397; 169. Analysis calculated for C₂₅ H₄₃O₁₀ N₅.H₂ CO₃ : C, 49.13; H, 7.14; N, 11.02%. Found: C, 49.10; H, 7.02;N, 11.38%.

B. Via Mixture of Cupric Acetate and Nickel (II) Acetate Complexes

(1) Add cupric acetate hydrate (8 gm., 40 mmol) and nickel (II) acetatetetrahydrate (10 gm., 40 mmol) to a stirred solution of sisomicin (8.94gm. 20 mmol) in dimethylsulfoxide (400 ml.). After 30 minutes at roomtemperature add dropwise a solution of acetic anhydride (5.4 ml. in 50ml. tetrahydrofuran, 60 mmol). After the addition is complete, stir thereaction mixture for an additional 30 minutes. Pour the reaction mixtureinto 1.5 l. of ether and thoroughly mix the contents. After letting themixture settle, decant the ether layer. Repeat the above procedure threetimes using 500 ml. ether each time. Dissolve the semi-solid residue in400 ml. methanol and bubble hydrogen sulfide through the solution toremove cupric and nickel (II) ions. Remove the solids by filtrationthrough Celite and wash the residue with methanol. Concentrate thecombined filtrates in vacuo and dissolve the residue in water (150 ml.).Treat the aqueous solution with Amberlite IRA-401S ion exchange resin inthe hydroxide cycle until the pH is about 9. Remove the resin byfiltration, wash thoroughly and either lyophilize the aqueous solutionto give 3,2',6'-tri-N-acetylsisomicin in 90% yield (10.3 gms.) orconcentrate the aqueous solution to a thick syrup, dissolve the residuein minimum amount of isopropanol and precipitate the product with excessether.

(2) Alternatively, after removal of the cupric and nickel (II) ions bytreatment with hydrogen sulfide and filtration, concentrate the filtrateand dissolve the residue in a minimum amount of isopropanol andprecipitate the product with excess ether to obtain3,2',6'-tri-N-acetylsisomicin acetic acid salt in 90% yield.

C. Via Cobalt (II) Acetate Complex

(1) Stir sisomicin (0.447 gms., 1 mmol) in dimethylformamide (20 ml.)and add cobalt (II) acetate tetrahydrate (0.516 gms., 2.07 mmol). Stirfor 20 minutes at room temperature, then to the cobalt (II) acetatecomplex thereby formed add dropwise a 1 molar solution of aceticanhydride in tetrahydrofuran (3 ml.). Continue stirring for 1 hour, thenadd water (10 ml.) and bubble hydrogen sulfide through the solutionuntil all the cobalt (II) is precipitated. Remove the cobalt (II)sulfide by filtration through a pad of Celite and wash the residue withwater. Evaporate the filtrate in vacuo and dissolve the resultantresidue in a minimum quantity of chloroform:methanol:ammonium hydroxide(2:1:0.35). Pass the solution through a column of silica gel (50 gms.,60-200 mesh), washing the column with the same solvent. Collect thehomogeneous fractions containing the 3,2',6'-tri-N-acetylsisomicin asdetermined by thin layer chromatography, evaporate the combinedfractions in vacuo and lyophilize the resultant residue to obtain3,2',6'-tri-N-acetylsisomicin (yield= 0.498 gms., 88% theory).

(2) In the above procedure, by substituting dimethylsulfoxide fordimethylformamide and by isolating and purifying the resultant productin a manner similar to that described in Example 1B, there is obtained3,2',6'-tri-N-acetylsisomicin in good yields.

D. Utilizing Acetyl-Acetone as Transition Metal Precipitating Reagent

In the procedures of Examples 1A, 1B and 1C in place of hydrogen sulfideutilize acetyl-acetone as the transition metal precipitating reagent andseparate by filtration the resultant precipitate of the correspondingtransition metal acetyl-acetonate, then isolate and purify each of theresultant products in a manner similar to that described to obtain3,2',6'-tri-N-acetylsisomicin.

EXAMPLE 2 3,2',6'-TRI-N-ACETYLVERDAMICIN VIA CUPRIC ACETATE COMPLEX

To a stirred solution of verdamicin (4.61 gms., 10 mmol) indimethylformamide (300 ml.) and water (90 ml.) add cupric acetatemonohydrate (14 gms., 70 mmol), stir the reaction mixture at roomtemperature for about 30 minutes, then to the cupric salt complexthereby formed add dropwise 33 ml. of a 1 molar solution of aceticanhydride in dimethylformamide (3.3 equivalents). Continue stirring atroom temperature for about 18-20 hours, then add water and bubblehydrogen sulfide gas through the solution. Filter off the copper sulfidethereby formed through filter aid on a sintered glass funnel rinsingwith water (50-200 ml.). Evaporate the filtrate in vacuo, dissolve theresultant residue in water and adjust the pH of the aqueous solution toabout 9 using Amberlite IRA-401S ion exchange resin in the hydroxideform. Freeze dry the solution to a residue comprising3,2',6'-tri-N-acetylverdamicin (5.5 gms.). Purify by chromatographing on300 gm. silica gel (60-200 mesh) eluting withchloroform:methanol:ammonium hydroxide (28%) (30:10:1). Combine likefractions as determined by thin layer chromatography and evaporate thecombined fractions in vacuo to a residue comprising3,2',6'-tri-N-acetylverdamicin, yield 1.68 gm. (2.8 mmol, 28% theory);pmr (ppm) (D₂ O): δ 1.25 (4"--C--CH₃); 1.36 (6'--C--CH₃, J_(6'),7' = 7Hz); 1.97, 2.04 (N-Acetyls); 2.56 (3"--N--CH₃); 4.91 (H-4"); 5.11 (H-1',J_(1'),2' = 4Hz); 5.60 (d, H-1", J_(1"),2" = 2.5 Hz); mass spectrum: m/e587 [M].sup..+ ; 392, 374, 364, 346; 233, 215, 205, 187, 457, 439, 429,411; 183.

EXAMPLE 3 3,2',6'-TRI-N-ACETYLAMINOGLYCOSIDES VIA CUPRIC ACETATE ANDNICKEL (II) ACETATE COMPLEXES A. 3,2',6'-Tri-N-Acetylaminoglycosides

In a manner similar to that described in Example 1B treat each of thefollowing aminoglycosides with cupric acetate hydrate and nickel (II)acetate tetrahydrate in dimethylsulfoxide followed by treatment of thesalt complex thereby formed with acetic anhydride in tetrahydrofuran,and thence treatment with hydrogen sulfide.

1. gentamicin C₁,

2. gentamicin C_(1a),

3. gentamicin C₂,

4. gentamicin C_(2a),

5. gentamicin C_(2b),

6. verdamicin,

7. tobramycin,

8. Antibiotic 66-40B,

9. antibiotic 66-40D,

10. antibiotic JI-20A,

11. antibiotic JI-20B,

12. antibiotic G-52,

13. 5-epi- and 5-epi-azido-5-deoxy analogs of the foregoing,

14. kanamycin B,

15. 3',4'-dideoxykanamycin B,

16. nebramycin factor 4,

17. nebramycin factor 5',

18. 3',4'-dideoxy-3',4'-dehydrokanamycin B,

19. 3',4'-dideoxy-6'-N-methylkanamycin B,

20. antibiotic Mu-2 (5-deoxysisomicin).

Isolate and purify each of the resultant products in a manner similar tothat described in Examples 1 and 2 to obtain the 3,2',6'-tri-N-acetylderivatives of each of the foregoing aminoglycosides.

EXAMPLE 4 3,2',6'-TRI-N-(2,2,2-TRICHLOROETHOXYCARBONYL) DERIVATIVES OFSISOMICIN AND GENTAMICIN C_(1a) VIA CUPRIC ACETATE COMPLEX A.3,2',6'-Tri-N-(2,2,2-Trichloroethoxycarbonyl)sisomicin

Add cupric acetate hydrate (14 gms., 70 mmol) to a stirred solution ofsisomicin (5.0 gms., 11.1 mmol) in dimethylsulfoxide (300 ml.) stirringfor 45 minutes, then to the cupric salt complex thereby formed add inportions N-(2,2,2-trichloroethoxycarbonyloxy)succinimide (9.25 gms., 32mmol) (over a 2 to 3 minute period). Stir the resulting green solutionfor one hour, then dilute the reaction mixture with 2 N ammoniumhydroxide (3 liters) and extract with ethyl acetate (two 600 ml.portions). Combine the organic extracts, wash with 2 N ammoniumhydroxide (600 ml.) containing sodium chloride (60 gms.), dry oversodium sulfate and evaporate in vacuo. Chromatograph the resultantresidue on silica gel (300-350 gms.) eluting with a chloroform:methanol:concentrated ammonium hydroxide solvent mixture (7:2:0.1 byvolume). Combine the like eluates containing the desired product asdetermined by thin layer chromatography and evaporate the combinedeluates in vacuo, then dry the resultant residue at 60° C. and 0.1 mm.to give 3,2',6'-tri-N-(2,2,2-trichloroethoxycarbonyl)sisomicin, yield7.52 gms. (77% theory); m.p. 124-127° C. [α]_(D) ²⁶ + 91.0° (c, 0.5 inchloroform); pmr (ppm) (CDCl₃): δ 1.17 (4"--C--CH₃), 2.58 (3"--N--CH₃)and 4.77 (--CH₂ CCl₃).

B. 3,2',6'-Tri-N-(2,2,2-Trichloroethoxycarbonyl)gentamicin C_(1a)

Add cupric acetate hydrate (2.8 gms.; 14 mmol) to a stirred solution ofgentamicin C_(1a) (1.0 gm., 2.22 mmol) in dimethylsulfoxide (56 ml.) at25° C. Continue stirring for 1 hour, then to the cupric salt complexthereby formed add portionwiseN-(2,2,2-trichloroethoxycarbonyloxy)succinimide (1.8 gms. 62 mmol) overa 15 minute period. Continue stirring for 2 hours, then dilute thereaction mixture with 2 N ammonium hydroxide (800 ml.) and extract withethyl acetate (3 × 75 ml.). Evaporate the combined extracts in vacuo andchromatograph the resultant residue on a silica gel column (110 × 2.5cm.) eluting first with chloroform (250 ml.) and then eluting withchloroform:methanol:concentrated ammonium hydroxide (7:2:0.1 by volume).Combine the like eluates containing the desired product as determined bythin layer chromatography and evaporate the combined eluates to aresidue of 3,2',6'-tri-N-(2,2,2-trichloroethoxycarbonyl)gentamicinC_(1a), yield 1.51 gms. (78% theory); [α] _(D) ²⁶ + 80.0° (c, 0.3 inchloroform), ν _(max) ^(KBr) 3330, 1730, 1520, 1040, 1025 cm⁻¹ ; pmr(ppm) (CDCl₃): δ 1.14 (4"--C--CH₃), 2.55 (3"--N--CH₃) and 4.64 (--CH₂CCl₃).

EXAMPLE 5 3,6'-DI-N-ACETYLAMINOGLYCOSIDES A.3,6'-Di-N-Acetylaminoglycosides

(1) In a manner similar to that described in Example 1A treat each ofthe following aminoglycosides with cupric acetate hydrate in aqueousdimethylformamide followed by treatment of the cupric salt complexthereby formed with acetic anhydride in dimethylformamide and thencereaction with hydrogen sulfide.

1. kanamycin A,

2. gentamicin B,

3. gentamicin B₁,

4. gentamicin B₂,

5. gentamicin A₃,

6. 6'-n-methylkanamycin A.

isolate and purify each of the resultant products in a manner similar tothat described in Example 1A to obtain the 3,6'-di-N-acetyl derivativesof each of the foregoing aminoglycosides.

(2) Alternatively, treat each of the aminoglycoside starting compoundsof above Example 5A(1) in a manner similar to that described in Example1B with cupric acetate hydrate, nickel (II) acetate tetrahydrate orcobalt (II) acetate tetrahydrate in dimethylsulfoxide followed bytreatment of the salt complex thereby formed with acetic anhydride intetrahydrofuran and thence reaction with hydrogen sulfide to obtain,upon isolation and purification in the described manner, thecorresponding 3,6'-di-N-acetyl derivatives of the startingaminoglycosides.

EXAMPLE 6 3,2'-DI-N-ACETYLAMINOGLYCOSIDES A.3,2'-Di-N-Acetylaminoglycosides

Treat each of the following aminoglycosides in a manner similar to thatdescribed in Example 5 A (1 and 2).

1. kanamycin C,

2. antibiotic G-418,

3. gentamicin A,

4. gentamicin A₁,

5. gentamicin X₂.

isolate and purify each of the resultant products in the describedmanner to obtain the corresponding 3,2'-di-N-acetyl derivatives of eachof the starting aminoglycosides.

EXAMPLE 7 2',6'-DI-N-SUBSTITUTED SISOMICIN A.2',6'-di-N-(2,2,2-Trichloroethoxycarbonyl)sisomicin Via Nickel (II)Acetate Complex

Add sisomicin (5 gms., 11.1 mmol) and nickel (II) acetate hydrate (15gms., 75 mmol) to methanol (350 ml.) and stir until dissolved. To thesolution of the nickel II salt complex thereby formed cooled in an icebath add N-(2,2,2-trichloroethoxycarbonyloxy)succinimide (6.40 gms., 22mmol) in portions over a 2 to 3 minute period and stir the reactionmixture at room temperature for an hour. Add concentrated ammoniumhydroxide (5 ml.) to the reaction mixture, evaporate in vacuo to avolume of about 100 ml., add 2 N ammonium hydroxide (500 ml.) togetherwith sodium chloride (50 gms.) and extract with chloroform (three 300ml. portions). Combine the organic phases, dry over sodium sulfate andevaporate. Chromatograph the resultant residue on 300 gms. of silica geleluting with a chloroform:methanol: concentrated ammonium hydroxidesolvent mixture (3:1:0.1 by volume). Combine like fractions containingthe desired product as determined by thin layer chromatography andevaporate the combined fractions in vacuo. Dry the precipitate at 60° C.at 0.1 mm. pressure to a residue of2',6'-di-N-(2,2,2-trichloroethoxycarbonyl)sisomicin, yield=5.80 gms.(72% theory); m.p. 115-119° C.; [α]_(D) ²⁶ + 94° C. (c, 0.4 inchloroform); pmr (ppm) (CDCl₃): δ 1.16 (4"--C--CH₃); 1.33 (H-2,J_(2ax),2eq =J₁,2ax =J_(2ax),3 =10 Hz); 2.60 (3"--N--CH₃); 4.73 (--CH₂CCl₃); 4.95 (H-1" and H-4'); 5.50 (H-1', H_(1'),2' =3 Hz); 6.28 (2'--NH,J=8 Hz) and 7.45 (6'--NH).

B. 2',6'-Di-N-Tert.-Butoxycarbonylsisomicin Via Cupric Acetate Complex

Add cupric acetate hydrate (0.6 gm., 3 mmol) to a solution of sisomicin(447 mg., 1 mmol) in dimethylsulfoxide (10 ml.), and stir for 10 minutesat room temperature. To the cupric salt complex thereby formed adddropwise a solution of N-tert.-butoxycarbonyloxyphthalimide (576 mg.,2.2 mmol) in dimethylsulfoxide (3 ml.). Stir for 18 hours at roomtemperature, then add the reaction solution dropwise into stirreddiethyl ether (75 ml.). Allow the resultant precipitate to settle,decant the diethyl ether solution and triturate the precipitate twicewith 75 ml. portions of diethyl ether. Dissolve the precipitate inmethanol, and bubble hydrogen sulfide through the methanol solution,separate the resultant cupric sulfide precipitate by filtration,deionize the methanolic solution with Amberlite IRA-401S (OH⊖) ionexchange resin (20 gm.), filter, concentrate the filtrate in vacuo andchromatograph the resultant residue on alumina (75 gms.) in a column(2.4 × 30 cm.) eluting with chloroform:methanol:concentrated ammoniumhydroxide (30:10:1 by volume). Combine the like fractions containing thedesired product as determined by thin layer chromatography and evaporatethe combined fractions and freeze dry the resultant residue to give2',6'-di-N-tert.-butoxycarbonylsisomicin, yield 258 mg. (40% theory);mass spectrum: (M⁺.) m/e 647, also m/e 547; 530; 517, 499, 489, 471;462; 350, 332, 322, 304; 191; 160.

C. 2',6'-Di-N-Tert.-Butoxycarbonylsisomicin Via Cobalt (II) AcetateComplex or Via Nickel (II) Acetate Complex

In the procedure of Example 7B, by utilizing methanol as solvent(instead of dimethylsulfoxide) and either nickel (II) acetate or cobalt(II) acetate as transition metal salt (instead of cupric acetate) thereis obtained 2',6'-di-N-tert.-butoxycarbonylsisomicin in improved yields.

EXAMPLE 8 1,3,2',6'-TETRA-N-ACETYLSISOMICIN VIA CUPRIC ACETATE COMPLEX

Add cupric acetate hydrate (49 gms., 24.5 mmol) to a stirred solution ofsisomicin (22 gms., 49.2 mmol) in 91% aqueous methanol. Continuestirring the solution in an ice water bath for 15 minutes, then to thecupric salt complex thereby formed add dropwise over a period of 10minutes 21 ml. of acetic anhydride (226 mmol) keeping the temperaturebelow 25° C. Stir the reaction mixture for 3 hours at room temperature,then bubble hydrogen sulfide through the solution. Remove the resultantprecipitate of cupric sulfide by filtration through Celite and wash theresidue with methanol. Combine the filtrate and washings and concentratein vacuo. Chromatograph the resultant residue on 600 gms. silica gel(60-200 mesh) eluting with a solvent mixture ofchloroform:methanol:concentrated ammonim hydroxide (30:10:1). Combinelike fractions containing the desired product as determined by thinlayer chromatography and evaporate in vacuo and lyophilize the resultantresidue to give 1,3,2',6'-tetra-N-acetylsisomicin, yield 23.5 gms. (78%theory); [α]_(D) ²⁶ + 191° (c, 0.2 in water); pmr (ppm) (D₂ O): δ 1.18(C--CH₃); 1.88-1.98 (N-acetyls); 2.48 (N--CH₃); 4.88 (H-4'); 5.07 (H-1",J_(1"),2" = 4 Hz); 5.48 (H-1', J_(1'),2' = 3 Hz); mass spectrum: (M⁺.)m/e 615; also m/e 592, 550; 485, 467, 457, 439; 434, 416, 406, 388; 275,257, 247, 229; 211; 160.

EXAMPLE 9 1,3,2'-TRI-N-ACETYLSISCOMICIN A. 6'-N-TrifluoroacetylsisomicinVia Cupric Acetate Complex

Add cupric acetate hydrate (17.48 gms., 87.3 mmol) to a stirred solutionof sisomicin (40 gms., 89.5 mmol) in 95% aqueous methanol (3.12 liters).Stir for 5 minutes, then to the cupric salt complex thereby formed adddropwise over a period of 3 minutes ethyl trifluorothiol acetate (11.65ml., 91.9 mmol). Continue stirring for 1 hour, then add an additionalportion of ethyl trifluorothiol acetate (1.6 ml., 12.45 mmol). Thisreaction solution containing 6'-N-trifluoroacetylsisomicin is used as isin the procedure of Example 9B.

B. 1,3,2'-Tri-N-Acetylsisomicin

Cool the reaction mixture prepared in Example 9A in an ice water bathand add dropwise acetic anhydride (28 gms., 280 mmol) at a rate of 25drops per minute. Stir for 18 hours at room temperature, then bubblehydrogen sulfide through the solution for 10 minutes. Remove theprecipitated cupric sulfide by filtration through a pad of Celite.Evaporate the filtrate in vacuo, dissolve the resultant residue inconcentrated ammonium hydroxide (1 liter) and stir for 3 hours at roomtemperature. Concentrate the solution in vacuo and chromatograph theresultant residue on 4.5 kilograms of silica gel (60-200 mesh) elutingwith a solvent system consisting of chloroform:methanol:concentratedammonium hydroxide (30:10:1). Combine the like fractions containing thedesired product as determined by thin layer chromatography, concentratethe combined fractions in vacuo and lyophilize the resultant residue togive 1,3,2'-tri-N-acetylsisomicin; [α]_(D) ²⁶ + 191.5° (c, 0.3 inwater); pmr (ppm) (D₂): δ 1.31 (C--CH₃); 1.92, 1.94, 1.96 (N-acetyls);2.82 (N--CH₃); 5.15 (H-1", J_(1"),2" = 3.5 Hz); 5.55 (H-1', J_(1'),2' =2.5 Hz); mass spectrum: (M⁺.) 573 also m/e 443, 425, 415, 397; 275, 257,247, 229; 434, 416, 406, 388; 169, 160.

EXAMPLE 10 6'-N-HYDROCARBONYLOXYCARBONYLAMINOGLYCOSIDES A.6'-N-Benzyloxycarbonylgentamicin B Via Cupric Chloride Complex

Add cupric chloride dihydrate (0.34 gms., 2 mmol) to a stirred solutionof gentamicin B (0.964 gms., 2 mmol) in water (3 ml.) anddimethylsulfoxide (30 ml.). Stir at room temperature for 20 minutes,then add dropwise to the cupric salt complex thereby formed in situ asolution of N-benzyloxycarbonyloxyphthalimide (1.105 gms., 4 mmol) indimethylsulfoxide (5 ml.). Monitor the progress of the reaction via thinlayer chromatography on silica gel using a solvent system consisting ofchloroform:methanol:concentrated ammonium hydroxide (2:1:0.35). When thereaction is complete as evidenced by thin layer chromatography (about 3hours) add water (10 ml.) to the reaction mixture and bubble hydrogensulfide through the solution. Remove the resultant precipitate of cupricsulfide by filtration through a pad of Celite and wash the residue withmethanol. Stir the combined filtrate methanol wash with AmberliteIRA-401S (OH.sup.⊖) ion exchange resin. Filter off the resin and washwith methanol. Evaporate the combined filtrate and methanol wash invacuo using benzene as a co-solvent until all the water is removed. Pourthe resulting concentrated reaction solution into a large volume ofmethylene chloride with stirring. Separate the resulting precipitate byfiltration and wash the precipitate with ether to obtain6'-N-benzyloxycarbonylgentamicin B; yield 100%; pmr (ppm) (D₂ O): δ 1.26(4"--C--CH₃); 2.65 (3"--N--CH₃); 5.13 and 7.43 (O--CH₂ --C₆ H₅).

B. In the procedure of above Example 10A substitute for gentamicin B anyaminoglycoside antibacterial agent having a primary carbinamine at C-5'to obtain the corresponding 6'-N-benzyloxycarbonylaminoglycoside, e.g.sisomicin, gentamicin C_(1a), gentamicin A, Antibiotic JI-20A,Antibiotic 66-40B, Antibiotic 66-40D, the 5-epi-, 5-epi-azido-5-deoxy-and 5-epi-amino-5-deoxy analogs of the foregoing, Antibiotic Mu-1,Antibiotic Mu-2, Antibiotic Mu-4, Antibiotic Mu-5, tobramycin, kanamycinA, kanamycin B, Antibiotic BBK-8, Antibiotic XK-88-3 and AntibioticXK-88-5.

C. In the procedure of Examples 10A and 10B by substituting forN-benzyloxycarbonyloxyphthalimide equivalent quantities of otherphthalimide reagents, e.g. N-tert.-butoxycarbonyloxyphthalimide orN-(2,2,2-trichloroethoxycarbonyloxy)phthalimide, there is obtained thecorresponding 6'-N-hydrocarbonyloxycarbonylaminoglycoside, e.g. thecorresponding 6'-N-tert.-butoxycarbonylaminoglycoside and6'-N-(2,2,2-trichloroethoxycarbonyl)-aminoglycoside derivatives.

D. 6'-N-Tert.-Butoxycarbonylgentamicin B Via Cupric Acetate Complex

Add cupric acetate hydrate (24 gms. 120 mmol) to a stirred solution ofgentamicin B (19.28 gms., 40 mmol) in dimethylsulfoxide (1 liter).Continue stirring for 20 minutes, to the cupric salt complex therebyformed in situ add a solution of N-tert.-butoxycarbonyloxyphthalimide(16 gms., 61 mmol) in dimethylsulfoxide (200 ml.) over a period of 20minutes. Stir for 18 hours at room temperature, then bubble hydrogensulfide through the solution to precipitate cupric sulfide. Remove thesolids by filtration through a pad of Celite and wash the residue with200 ml. of water. Stir the combined filtrate and washings with 200 ml.of Amberlite IRA-401S (OH.sup.⊖) ion exchange resin for one hour. Removethe resin by filtration, wash with water, and concentrate the combinedfiltrate and washings in vacuo using benzene to azeotrope with water.Dissolve the resultant residue in methanol and pour the methanolsolution into excess ether with stirring. Filter and air dry theresultant precipitate comprising 6'-N-tert.-butoxycarbonylgentamicin B,yield 23 gm. (100% theory); [α]_(D) ²⁶ + 124° (c, 1 in methanol). pmr(ppm) (D₂ O): δ 1.21 (4"--C--CH₃); 1.42 (t-butyl); 2.55 (3"--N--CH₃);5.06 (H-1", J_(1"),2" = 4.0 Hz); 5.21 (H-1', J_(1'),2' = 3.0 Hz).Analysis Calculated for C₂₄ H₄₆ N₄ O₁₂.CO₂.H₂ O: C, 46.57; H, 7.51; N,8.68%. Found: C, 46.80; H, 7.82; N, 8.54%.

EXAMPLE 11 3,6'-DI-N-HYDROCARBONYLOXYCARBONYL AMINOGLYCOSIDES3,6'-Di-N-Benzyloxycarbonylgentamicin B via Mixture of Cupric Acetateand Nickel (II) Acetate Complexes

Add cupric acetate hydrate (8 gms., 40 mmol) and nickel (II) acetatetetrahydrate (9.92 gms., 40 mmol) to a stirred solution of gentamicin B(9.64 gms., 20 mmol) in dimethylsulfoxide (400 ml.). Stir at roomtemperature for 30 minutes, then to the cupric-nickel (II) salt complexthereby formed add N-benzyloxycarbonyloxyphthalimide (14 gms., 47.2mmol) in dimethylsulfoxide (70 ml.) dropwise over a 10 minute period.Stir for one hour at room temperature, then pour the reaction mixtureinto ether (4 l.) and shake for one minute. Allow the oil to settle anddecant off the supernatant ether. Repeat this procedure two more timesusing 1500 ml. and 1000 ml., respectively, of diethyl ether. Dissolvethe resultant gummy residue thereby obtained in methanol (400 ml.) andconcentrated ammonium hydroxide (40 ml.) and bubble hydrogen sulfidethrough the solution, separate the resultant precipitate comprisingcupric sulfide and nickel sulfide by filtration through a pad of Celite.Wash the residue with methanol, then stir the combined filtrate andmethanol wash with Amberlite IRA-401S (OH.sup.⊖) ion exchange resin (400ml.) to remove the N-hydroxy phthalimide. Filter the solution, wash theresin with methanol, then evaporate the combined filtrate and methanolwash in vacuo, and chromatograph the resultant residue on silica gel(900 gms.) eluting with chloroform:methanol:concentrated ammoniumhydroxide (30:10:1). Combine the like fractions containing the desiredproduct as determined by thin layer chromatography and evaporate thecombined fractions in vacuo to a residue comprising the3,6'-di-N-benzyloxycarbonylgentamicin B; yield 10.86 gms. (75% theory);[α]_(D) ²⁶ + 105.3° (c, 4.07 in water).

Analysis Calculated for: C₃₅ H₅₀ O₁₄ N₄.CO₂.2H₂ O: C, 52.04; H, 6.55; N,6.74%. Found: C, 51.94; H, 6.33; N, 6.83%.

B. 3,6'-di-N-Benzyloxycarbonylgentamicin B Via Cobalt (II) AcetateComplex

Dissolve gentamicin B (4.82 gms., 10 mmol) in dimethylsulfoxide (195ml.) and water (5 ml.). Add triethylamine (2 ml.) and stir. Add cobalt(II) acetate tetrahydrate (7.08 gms., 28.5 mmol), continue stirring for30 minutes, then to the gentamicin B-cobalt (II) acetate complex therebyformed add in a dropwise manner a solution ofN-(benzyloxycarbonyloxy)phthalimide (6.5 gms., 20 mmol) indimethylsulfoxide (20 ml.) and stir for 3 hours, then pour into etherand isolate in the manner described in Example 11A. Purify the resultantresidue by dissolving in a small amount of methanol and adding ethylacetate followed by excess diethyl ether. Separate the resultantprecipitate by filtration and air dry to obtain pure3,6'-di-N-benzyloxycarbonylgentamicin B, yield 90% theory.

C. 3,6'-di-N-Benzyloxycarbonylgentamicin B Via Cadmium (II) AcetateComplex

Dissolve gentamicin B (4.82 gms., 10 mmol) in dimethylsulfoxide (195ml.) and add cadmium (II) acetate dihydrate (7.98 gms., 30 mmol). Stirfor 30 minutes, then to the gentamicin B cadmium (II) acetate complexthereby formed add a solution of N-(benzyloxycarbonyloxy)phthalimide(6.5 gms., 20 mmol) in dimethylsulfoxide (20 ml.). Stir for 3 hours atroom temperature, then isolate and purify in a manner similar to thatdescribed in Example 11A to obtain 3,6'-di-N-benzyloxycarbonylgentamicinB.

D. 3,6'-Di-N-Benzyloxycarbonylkanamycin A Via Nickel (II) AcetateComplex

Add nickel (II) acetate tetrahydrate (24.8 gms., 10 mmol) to a stirredsolution of kanamycin A (9.7 gms., 20 mmol) in dimethylsulfoxide (400ml.). Stir at room temperature for 30 minutes, then to the resultingnickel (II) salt complex thereby formed addN-benzyloxycarbonyloxyphthalimide (13.0-15.6 gms., 44-52.6 mmol) indimethylsulfoxide (70 ml.) over a 10 minute period. Stir at roomtemperature for one hour, then pour the reaction mixture into ether(2500 ml.) and shake one minute. Allow the oil to settle and decant thesupernatant dimethylsulfoxide ether. Repeat this procedure two moretimes using 1500 ml. and 1000 ml., respectively, of ether. Dissolve theresultant gummy residue in methanol (1000 ml.) and concentrated ammoniumhydroxide (50 ml.). Bubble hydrogen sulfide through the solution andseparate the resultant precipitate of nickel (II) sulfide through a padof Celite and wash the residue with methanol. Stir the combined filtrateand methanol wash with Amberlite IRA-401S (OH.sup.⊖) ion exchange resin(300 gms.), filter off the resin and evaporate the filtrate in vacuo.Triturate the resultant gummy residue with a 50:50 mixture ofacetonitrile:ether and filter the resultant white solid to obtain3,6'-di-N-benzyloxycarbonylkanamycin A; yield 12.5 gms. (83.5% theory);[α]_(D) ²⁶ + 78° (c, 4.84 in 50% aqueous methanol). Analysis Calculatedfor: C₃₄ H₄₈ O₁₅ N₄.H.sub. 2 O; C, 56.2; H, 6.75; N, 6.94%. Found: C,51.02; H, 6.26; N, 6.85%.

E. Treat each of the following aminoglycosides with a mixture of cupricacetate hydrate and nickel (II) acetate tetrahydrate or with cobalt (II)acetate tetrahydrate or with cadmium (II) acetate dihydrate according tothe procedures of Examples 11A through 11D, respectively.

1. gentamicin B₁,

2. gentamicin A₃,

3. 6'-n-methylkanamycin A.

isolate and purify each of the resulting products to obtain,respectively,

1. 3,6'-di-N-benzyloxycarbonylgentamicin B₁,

2. 3,6'-di-N-benzyloxycarbonylgentamicin A₃,

3. 3,6'-di-N-benzyloxycarbonyl-6'-N-methylkanamycin A.

F. Preparation of 3,6'-Di-N-Tert.-Butoxycarbonylgentamicin B from6'-Tert.-Butoxycarbonylgentamicin B Via Cupric Acetate Complex

To a solution of 6'-N-tert.-butoxycarbonylgentamicin B (10 gms., 17.1mmol) in dimethylsulfoxide (300 ml.), add cupric acetate monohydrate(6.9 gms., 34.5 mmol) and stir for 20 minutes. To the cupric saltcomplex thereby formed in situ add dropwise a solution ofN-tert.-butoxycarbonyloxyphthalimide (4.5 gms., 17.1 mmol) indimethylsulfoxide (20 ml.). Continue stirring for 2 hours, then add anadditional 2.4 gms. of N-tert.-butoxycarbonyloxyphthalimide and continuestirring the reaction mixture for 10 hours. Pour the reaction mixtureinto ether (1.5 liters) with stirring. Allow the resultant precipitateor oil to settle and carefully decant the supernatant liquid. Wash theresidue two times with ether (300 ml.). Dissolve the resultant residuein methanol (100 ml.) and bubble hydrogen sulfide through the solutionto precipitate cupric sulfide completely. Remove the solids byfiltration through a pad of Celite and wash with methanol. Stir themethanolic solution with just enough Amberlite IRA-401S (OH.sup.⊖) ionexchange resin to bring the pH to about 9 and removeN-hydroxyphthalimide. Remove the resin by filtration, wash withmethanol, and concentrate the combined filtrate and washings to a volumeof about 50 ml. containing 3,6'-di-N-tert.-butoxycarbonylgentamicin B,which may be used without further purification as starting compound inthe procedure of Examples 18A and 21B. To isolate the compound,crystallize by triturating the residue after evaporation with ether togive 3,6'-di-N-tert.-butoxycarbonylgentamicin B (yield = 90% theory).Alternatively, purify by chromatographing on silica gel (ratio ofcompound: silica gel = 1:40) using chloroform:methanol:ammoniumhydroxide (2:1:0.35) as the developing solvent mixture. Pool the likefractions containing 3,6'-di-N-tert.-butoxycarbonylgentamicin B asdetermined by thin layer chromatography, concentrate and lyophilize togive 3,6'-di-N-tert.-butoxycarbonylgentamicin B; [α]_(D) ²⁶ + 113.3° (c,0.39 in water); pmr (ppm) (D₂ O): δ 1.17 (4"--C--CH₃); 1.38 (t-butyl);2.5 (3"--N--CH₃); 2.54 (H-3", J_(2"),3" = 10 Hz); 4.02 (H-5" eq, J_(5")ax,eq = 12 Hz); 5.04 (H-1", J_(1"),2" = 3.5 Hz); 5.23 (H-1', J_(1'),2' =3.0 Hz).

Analysis calculated for C₂₉ H₅₇ O₁₅ N₄.H₂ O: C, 49.63; H, 8.19; N,7.98%. Found: C, 49.83; H, 7.81; N, 7.68%.

G. 3,6'-Di-N-Tert.-Butoxycarbonylgentamicin B from Gentamicin B ViaCupric Acetate Complex

Add cupric acetate monohydrate (5.0 gms., 25 mmol) to a stirred solutionof gentamicin B (4.82 gms., 10 mmol) in dimethylsulfoxide (250 ml.).Stir for 30 minutes, then to the cupric salt complex thereby formed insitu add dropwise a solution of N-tert.-butoxycarbonyloxyphthalimide(10.5 gms., 40 mmol) in dimethylsulfoxide (20 ml.). Stir at roomtemperature for 2 hours, then add additionalN-tert.-butoxycarbonyloxyphthalimide (1.5 gms., 5.7 mmol). Stir for 16hours, then again add an additional N-tert.-butoxycarbonyloxyphthalimide(0.35 gms., 1.33 mmol). Continue stirring for 6 hours, then pour thereaction mixture into 1.5 l of stirred ether. Stir for 30 minutes, allowthe reaction mixture to stand, decant the supernatant solution, againadd 500 ml. of ether to the residue, let stand and decant thesupernatant solution. Dissolve the resultant residue in 100 ml. ofmethanol and bubble hydrogen sulfide for 15 minutes. Remove the cupricsulfide solid by filtration through a pad of Celite and wash the residuewith methanol. Treat the methanolic solution with Amberlite IRA-401S(OH.sup.⊖) ion exchange resin to bring the pH of the solution to about8.5-9.0, remove the solids by filtration and wash the solids withmethanol. Evaporate the solution to near dryness and lyophilize fromwater. Crystallize 3,6'-di-N-tert.-butoxycarbonylgentamicin B bytriturating with ether. Alternatively, the crude concentrate may be usedas starting material in the procedure of Examples 18A and 21B.

EXAMPLE 12 OTHER 3,2',6'-TRI-N-ACYLAMINOGLYCOSIDES A.3,2',6'-Tri-N-Benzyloxycarbonylgentamicin C₂ Via Mixture of CupricAcetate Complex and Nickel (II) Acetate Complex

Add cupric acetate hydrate (0.6 gms., 3 mmol) and nickel (II) acetatetetrahydrate (0.743 gms., 3 mmol) to a stirred solution of gentamicin C₂(0.925 gms., 2 mmol) in dimethylsulfoxide (40 ml.). Stir at roomtemperature for 30 minutes, then to the mixed cupric and nickel saltcomplex thereby formed add N-benzyloxycarbonyloxyphthalimide (1.8 gms.,6.5 mmol) in dimethylsulfoxide (7 ml.) over a 10 minute period. Continuestirring for one hour, then pour the reaction mixture into ether (400ml.) with stirring. Decant the supernatant ether, repeat the foregoingprocess using 200 ml. and 100 ml. of ether, respectively. Dissolve theresulting gummy residue in methanol (100 ml.) and concentrated ammoniumhydroxide (5 ml.), then bubble hydrogen sulfide through the solution.Remove the resultant precipitate of cupric sulfide and nickel (II)sulfide by filtration through a pad of Celite, wash the precipitate withmethanol then stir the combined filtrate and methanol wash withAmberlite IRA-401S (OH.sup.⊖) ion exchange resin. Separate the resin byfiltration and wash with methanol. Evaporate the combined filtrate andmethanol wash in vacuo, then chromatograph the resultant gummy residueon silica gel (50 gms.) eluting with chloroform:methanol:concentratedammonium hydroxide (30:10:1). Combine the like fractions containing thedesired product as determined by thin layer chromatography and evaporatethe combined eluates to a residue comprising3,2',6'-tri-N-benzyloxycarbonylgentamicin C₂ ; yield 1.4 gms. (81%theory): mass spectrum: m/e 484; 411; 325 160.

B. In a manner similar to that described in Example 12A, treat each ofthe aminoglycoside starting compounds (1), (2) and (4)-(20) listed inExample 3A with cupric acetate hydrate and nickel (II) acetatetetrahydrate in dimethylsulfoxide followed by treatment of the saltcomplex thereby formed with N-benzyloxycarbonyloxyphthalimide and thencetreatment with hydrogen sulfide and isolation and purification of eachof the resultant products to obtain, respectively,

1. 3,2',6'-tri-N-benzyloxycarbonylgentamicin C₁, yield 1.3 gms., 72.5%theory. Mass spectrum: m/e 749; 615; 484, 466, 456; 425, 407; 325, 307,297; 291; 160.

2. 3,2',6'-tri-N-benzyloxycarbonylgentamicin C_(1a),

4. 3,2',6'-tri-N-benzyloxycarbonylgentamicin C_(2a),

5. 3,2',6'-tri-N-benzyloxycarbonylgentamicin C_(2b),

6. 3,2',6'-tri-N-benzyloxycarbonylverdamicin,

7. 3,2',6'-tri-N-benzyloxycarbonyltobramycin,

8. 3,2',6'-tri-N-benzyloxycarbonyl-Antibiotic 66-40B,

9. 3,2',6'-tri-N-benzyloxycarbonyl-Antibiotic 66-40D,

10. 3,2',6'-tri-N-benzyloxycarbonyl-Antibiotic JI-20A,

11. 3,2',6'-tri-N-benzyloxycarbonyl-Antibiotic JI-20B,

12. 3,2',6'-tri-N-benzyloxycarbonyl-Antibiotic G-52,

13. 5-epi- and 5-epi-azido-5-deoxy analogs of the foregoing,

14. 3,2',6'-tri-N-benzyloxycarbonylkanamycin B,

15. 3,2',6'-tri-N-benzyloxycarbonyl-3',4'-dideoxykanamycin B,

16. 3,2',6'-tri-N-benzyloxycarbonylnebramycin factor 4,

17. 3,2',6'-tri-N-benzyloxycarbonylnebramycin factor 5',

18. 3,2',6'-tri-N-benzyloxycarbonyl-3',4'-dideoxy-3',4'-dehydrokanamycinB,

19. 3,2',6'-tri-N-benzyloxycarbonyl-3',4'-dideoxy-6'-N-methylkanamycinA,

20. 3,2',6'-tri-N-benzyloxycarbonyl-Antibiotic Mu-2.

C. In the procedures of Examples 12A and 12B, substitute for theN-benzyloxycarbonyloxyphthalimide reagent equivalent quantities ofN-tert.-butoxycarbonyloxyphthalimide orN-(2,2,2-trichloroethoxycarbonyloxy)succinimide to obtain thecorresponding 3,2',6'-tri-N-tert.-butoxycarbonylaminoglycoside and3,2',6'-tri-N-(2,2,2-trichloroethoxycarbonyl)aminoglycoside,respectively.

D. Mixed 3,2',6'-Tri-N-Acylaminoglycosides and Conversion to3,6'-Di-N-Acylaminoglycoside (1)6'-N-Acetyl-2'-N-(2,2,2-Trichloroethoxycarbonyl)sisomicin Via CupricAcetate Complex

In a manner similar to that described in Example 7A treat6'-N-acetylsisomicin with about 7 equivalents of cupric acetate hydratein methanol, then to the cupric salt complex thereby formed add inportions about 1 equivalent ofN-(2,2,2-trichloroethoxycarbonyl)succinimide. Stir the reaction mixturefor one hour, then treat with ammonium hydroxide, and isolate and purifythe resultant product in a manner similar to that described in Example7A to obtain 2'-N-(2,2,2-trichloroethoxycarbonyl)-6'-N-acetylsisomicin.

(2) 3,6'-Di-N-Acetyl-2'-N-(2,2,2-Trichloroethoxycarbonyl)sisomicin ViaCupric Acetate Complex

Add cupric acetate hydrate (24 gms., 120 mmol) to a stirred solution of2'-N-(2,2,2-trichloroethoxycarbonyl)-6'-N-acetylsisomicin (26.5 gms., 40mmol) in dimethylsulfoxide (1 liter). Continue stirring for 20 minutes,then to the cupric salt complex thereby formed add dropwise at the rateof about 25 drops per minute 40 ml. of a 1 molar solution of aceticanhydride in tetrahydrofuran (40 mmol). Stir the reaction mixture for anadditional 30 minutes, then pour into ether (8 l.). Shake well and setaside. Decant the ether layer and wash the residue twice more with 1liter ether each time. Dissolve the residue in methanol (800 ml.) andbubble hydrogen sulfide for 15 minutes. Stir the mixture for anadditional 30 minutes, then filter the solution through a pad of Celiteand wash the cupric sulfide residue with water. Concentrate the combinedfiltrate and water washings and chromatograph the resultant residue onsilica gel eluting with chloroform:methanol:ammonium hydroxide(30:10:1). Combine like fractions containing the desired product asdetermined by thin layer chromatography and evaporate the combinedfractions in vacuo to a residue comprising3,6'-di-N-acetyl-2'-N-(2,2,2-trichloroethoxycarbonyl)sisomicin.

(3) 3,6'-Di-N-Acetylsisomicin

Add 3.9 molar equivalents of zinc powder to a solution of3,6'-di-N-acetyl-2'-N-(2,2,2-trichloroethoxycarbonyl)sisomicin in 10%acetic acid in methanol. Heat the solution at reflux temperature for twohours monitoring the reaction by thin layer chromatography on silica gelusing chloroform:methanol:ammonium hydroxide (30:10:1) as solventsystem. When the reaction is complete as determined by thin layerchromatography, filter the solution, add sodium carbonate to thefiltrate, filter and concentrate the filtrate in vacuo. Purify bychromatographing the resultant residue on silica gel eluting withchloroform:methanol:ammonium hydroxide (30:10:1). Combine the likefractions containing the desired product as determined by thin layerchromatography, then evaporate the combined fractions in vacuo andlyophilize the resultant aqueous mixture to a residue comprising3,6'-di-N-acetylsisomicin.

EXAMPLE 13 1,3,2',3"-TETRA-N-ACETYLSISOMICIN VIA CUPRIC ACETATE COMPLEXA. 6'-N-Trifluoroacetylsisomicin Via Cupric Acetate Complex

Dissolve sisomicin (39.1 gms., 87.5 mmol) in methanol (2950 ml.) andwater (200 ml.). While stirring add cupric acetate hydrate (8.92 gms.,44.7 mmol). Stir for 15 minutes, then add dropwise over a period ofthree minutes ethyl trifluorothiolacetate (11.55 ml., 14.2 gms., 90mmol) followed by an additional 1.3 ml. (1.6 gms., 10 mmol) of ethyltrifluorothiolacetate. Remove the methanol by evaporation in vacuo, thenadd water until the reaction volume is 300 ml. Pass hydrogen sulfidethrough the reaction solution, then filter the resultant cupric sulfideprecipitate through a Celite pad to obtain a solution containing6'-N-trifluoroacetylsisomicin, which is used as is in the procedure ofExample 13B.

B. 1,3,2',3"-Tetra-N-Acetyl-6'-N-Trifluoroacetylsisomicin

To the solution obtained in Example 13A add 300 ml. of methanol, thenadd acetic anhydride (109 ml., 1.16 Mol, 13.2 equivalents) adjusting thepH of the reaction mixture to 8.5 with triethylamine prior to adding thelast 10 ml. of acetic anhydride. Stir for 18 hours at room temperature,then evaporate in vacuo to a residue comprising1,3,2',3"-tetra-N-acetyl-6'-N-trifluoroacetylsisomicin.

C. 1,3,2',3"-Tetra-N-Acetylsisomicin

Dissolve the 1,3,2',3"-tetra-N-acetyl-6'-N-trifluoroacetylsisomicinprepared in Example 12B in 500 ml. of concentrated ammonium hydroxide.Stir at room temperature overnight and evaporate in vacuo. Dissolve theresultant residue in water and stir the solution with Amberlite IRA-401S(OH.sup.⊖) ion exchange resin. Remove the resin by filtration, wash withmethanol, then evaporate the combined filtrate and methanol wash invacuo followed by chromatography of the resultant residue on a 1.7kilogram silica gel column (7.5 × 165 cm.) eluting with the lower phaseof a chloroform:methanol:ammonium hydroxide (28%) (2:1:1) solventsystem. Combine the like fractions containing the desired product asdetermined by thin layer chromatography and evaporate the combinedeluates to a residue comprising 1,3,2',3"-tetra-N-acetylsisomicin, yield29.9 gms., (55% theory); [α]_(D) ²⁶ + 207.4° (c, 0.3 in water); pmr(ppm) (D₂ O): δ 1.07, 1.17 (4"--C--CH₃); 1.95, 1.98, 2.03 (N-acetyls);3.13, 3.00 (3"--N--CH₃); 5.29 (H-1", J_(1"),2" = 4.0Hz); 5.64 (H-1',J_(1'),2' = 2.5Hz); mass spectrum: (M⁺.) m/e 615, also m/e 598; 592;550; 443, 425, 415, 397; 453, 411; 275, 257, 247, 229; 202; 169.Analysis calculated for: C₂₇ H.sub. 45 O₁₁ N₅.1.5 H₂ O: C, 50.43; H,7.52; N, 10.89%. Found: C, 50.61; H, 7.36; N, 10.79%.

EXAMPLE 14 ISOLATION OF AMINOCYCLITOL-AMINOGLYCOSIDE TRANSITION METALSALT COMPLEXES A. Isolation of Gentamicin B-dicupric acetate Complex

Add cupric acetate hydrate (0.4 gms., 2 mmol) to a solution ofgentamicin B (0.482 gms., 1 mmol) in methanol (25 ml.), stir for 30minutes at room temperature then evaporate the solvent in vacuo and drythe resultant residue at 60° C. in vacuo to obtain gentamicin B-dicupricacetate complex as a deep blue colored solid (in contrast to greencolored cupric acetate dihydrate); yield 0.8 gms.; λ_(max) ^(nujol)6.42, 8.72, 13.90 mμ.

B. Isolation of Gentamicin B-dicobalt (II) Acetate Complex

(1) Dissolve gentamicin B (0.482 gms., 1 mmol) in dimethylsulfoxide (20ml.) and add cobalt (II) acetate tetrahydrate (0.598 gms., 2 mmol). Stirfor 30 minutes at room temperature and then pour into ether (30 ml.).Decant the ether layer and triturate the residue with fresh ether (20ml.). Repeat this process once more, then triturate with ethyl ether.Isolate the resultant solid by filtration, wash with ethyl ether and dryto obtain gentamicin B-dicobalt (II) acetate complex as a lavendercolored solid (in contrast to pink colored cobalt (II) acetatetetrahydrate), yield 0.8 gms. λ_(max) ^(nujol) 5.88, 6.36, 10.59, 12.42,12.92, 13.90 μ.

(2) Dissolve the above isolated gentamicin B-dicobalt (II) acetatecomplex in dimethylsulfoxide and treat withN-(benzyloxycarbonyloxy)succinimide in a manner similar to thatdescribed in Example 11B followed by isolation and purification in thedescribed manner to obtain 3,6'-di-N-benzyloxycarbonylgentamicin B. C.In a manner similar to that described in Examples 14A and 14B, treatgentamicin B with nickel (II) acetate tetrahydrate and with cadmiun (II)acetate dihydrate and isolate and purify the complexes thereby formed toobtain gentamicin B-dinickel (II) acetate complex and gentamicinB-dicadmium (II) acetate complex.

In similar manner each of the aminocyclitol-aminoglycoside transitionmetal salt complexes prepared in situ in the preceding Examples may beisolated.

EXAMPLE 15 REACTION OF 1,3-DI-DE-N-AMIDINODIHYDROSTREPTOMYCIN WITHN-BENZYLOXYCARBONYLOXYPHTHALIMIDE WITH AND WITHOUT CUPRIC ACETATECOMPLEX A. 1,3-Di-De-N-Amidinodihydrostreptomycin

Add a solution of dihydrostreptomycin sulfate (25 gms., 17.1 mmol) inwater (100 ml.) to a solution of barium hydroxide (54 gms.) dissolved inwater (600 ml.). Separate the barium sulfate thereby formed byfiltration and heat the filtrate at reflux temperature under anatmosphere of nitrogen for 62 hours. To the cooled reaction mixture add4 N sulfuric acid dropwise until the reaction mixture reaches a pH of 7.Separate the barium sulfate thereby formed by filtration, add AmberliteIRA-401S (OH.sup.⊖) resin to the filtrate and heat the mixture until thefiltrate reaches a pH of 10. Remove the water by distillation andchromatograph the resultant residue on silica gel (600 gms.) elutingwith a solvent mixture of chloroform/methanol/28% ammonium hydroxide(3:4:2). Collect the like fractions containing1,3-di-de-N-amidinodihydrostreptomycin as determined by thin layerchromatography and evaporate the combined eluates to a residue of1,3-di-de-N-amidinodihydrostreptomycin (yield -- 10.9 gms.).

B. Reaction of 1,3-Di-De-N-Amidinodihydrostreptomycin Via Cupric AcetateComplex with N-Benzyloxycarbonyloxyphthalimide to Produce2"-N-Benzyloxycarbonyl-1,3-di-de-N-Amidinodihydrostreptomycin

To a solution of 1,3-di-de-N-amidinodihydrostreptomycin (5 gms., 10mmol) in dimethylsulfoxide (200 ml.) add cupric acetate monohydrate (2gms., 10 mmol) and stir at room temperature for 15 minutes, then to thecupric salt complex thereby formed add dropwise with continuous stirringof the reaction mixture N-benzyloxycarbonyloxyphthalimide (5.5 gms.) indimethylsulfoxide (20 ml.). Continue stirring for 2 hours, then pour thereaction mixture into diethyl ether (2 liters). Decant the supernatantether and repeat the foregoing process using 1 liter and 500 ml. ofdiethyl ether, respectively. Dissolve the resultant gummy residue in asolvent mixture comprising methanol (150 ml.) and 28% ammonium hydroxide(10 ml.). Bubble hydrogen sulfide through the solution and separate theresultant precipitate of cupric sulfide by filtration through a pad ofCelite, evaporate the filtrate and chromatograph the resultant residueon silica gel (400 gms.) eluting with a solvent mixture ofchloroform/methanol/28% ammonium hydroxide (2:1:0.35). Combine the likeeluates containing the desired compound as determined by thin layerchromatography and concentrate the combined eluates to a residuecomprising2"-N-benzyloxycarbonyl-1,3-di-de-N-amidinodihydrostreptomycin, yield 6gms. (98% theory); [α]_(D) ²⁶ -96.5° (c, 0.5 in water). pmr (ppm) (D₂O): δ 1.21 (d, 4'--C--CH₃, J=6 Hz); 3.04 (s, 2"--N--CH₃); 5.13 (s, C₆ H₅CH₂ OCONH); 7.41 (s, C₆ H₅ CH₂ OCONH); Analysis Calculated for: C₂₇ H₄₃O₁₄ N₃ ·H₂ O; Calc.: C, 49.76; H, 6.96; N, 6.45. Found: C, 49.69; H,6.78; N, 6.27. CD: [θ]_(TACu) ²⁸⁵ = +7,320.

C. Reaction of 1,3-Di-De-N-Amidinodihydrostreptomycin withN-Benzyloxycarbonyloxyphthalimide Without Cupric Acetate Complex Wherebyis Produced 1-N-Benzyloxycarbonyl-1,3-Di-De-N-Amidinodihydrostreptomycin

To a solution of 1,3-di-de-N-amidinodihydrostreptomycin (1 gm., 2.0mmol) in N,N-dimethylformamide (40 ml.) add a solution ofN-benzyloxycarbonyloxyphthalimide (0.8 gms., 2.7 mmol) inN,N-dimethylformamide (4 ml.). Stir the reaction mixture for 1 hour,then add water (10 ml.) and Amberlite IRA-401S (OH.sup.⊖) resin untilthe pH of the reaction mixture reaches 8.5. Separate the resin byfiltration, remove the solvents by distillation, then chromatograph theresultant residue on silica gel (100 gms.) eluting with a solventmixture of chloroform/methanol/28% ammonium hydroxide (2:1:0.35).Collect the like eluates containing1-N-benzyloxycarbonyl-1,3-di-de-N-amidinodihydrostreptomycin asdetermined by thin layer chromatography and evaporate the combinedeluates to a residue of1-N-benzyloxycarbonyl-1,3-di-de-N-amidinodihydrostreptomycin, yield0.417 gms. (33% theory); [α]_(D) ²⁶ -93.6° (c, 0.55% in water). pmr(ppm) (D₂ O): δ 1.22 (d, 4'--C--CH₃, J=6 Hz); 2.42 (s, 2" --N--CH₃);5.12 (s, C₆ H₅ CH₂ OCONH); 7.40 (s, C₆ H₅ CH₂ OCONH); Analysiscalculated for: C₂₇ H₄₃ O₁₄ N₃ ·H₂ O; Calc.: C, 49.76; H, 6.96; N, 6.45.Found: C, 49.41; H, 6.55; N, 6.21. CD: [θ]_(TACu) ²⁸² = +11,100.

PREPARATION OF 1-N-ALKYLAMINOGLYCOSIDES USING SELECTIVELY BLOCKEDINTERMEDIATES EXAMPLE 16 CONVERSION OF 3,2',6'-TRI-N-ACETYLSISOMICIN TO1-N-ETHYLSISOMICIN A. 3,2',6'-Tri-N-Acetyl-1-N-Ethylsisomicin

Add 0.1 N hydrochloric acid to a stirred solution of3,2',6'-tri-N-acetylsisomicin (1.146 gms., 2 mmol) in 20 ml. of water soas to bring the pH of the solution of 2.7. Cool the reaction mixture to3° C. and add 2.2 ml. of a 1 molar solution of acetaldehyde intetrahydrofuran (2.2 mmol), then add dropwise a solution of sodiumcyanoborohydride (0.16 gms., 2.6 mmol) in 2 ml. of water. Stir thereaction mixture for one hour maintaining the temperature below 5° C.and the pH at about 2.7 by the addition of 0.1 N hydrochloric acid, thenadd 0.44 ml. of a 1 molar solution of acetaldehyde in tetrahydrofuran(0.44 mmol) followed by a solution of sodium cyanoborohydride (35 mg.,0.56 mmol) in a few drops of water, maintaining the pH at 2.7 by theaddition of 0.1 N hydrochloric acid. Continue stirring the reactionmixture for one hour and repeat 2 times the foregoing addition of 0.22ml. of acetaldehyde solution and 10 mg. of sodium cyanoborohydride.Continue stirring at room temperature for 18 hours, then bring thesolution to a pH of 9 by stirring the solution with Amberlite IRA-401Sion exchange resin in the hydroxide form. Remove the resin byfiltration, wash with water and concentrate the combined filtrate andwashings in vacuo, and chromatograph the resultant residue on 100 gms.of silica gel (60-200 mesh) eluting with chloroform:methanol:ammoniumhydroxide (30:10:0.2). Combine the like fractions as determined by thinlayer chromatography on silica gel or using the same solvent system asabove but in the proportion of 30:10:1 and concentrate in vacuo thecombined eluates containing the major product and lyophilize theresultant aqueous mixture to a residue comprising3,2',6'-tri-N-acetyl-1-N-ethylsisomicin, yield 0.84 gms. (70% theory);[α]_(D) ²⁶ + 165° (c, 0.8 in water); pmr (ppm) (D₂ O): δ 1.05 (CH₂ CH₃,J=7.0 Hz); 1.17 (4"--C--CH₃); 1.84, 1.9, 1.91 (N-acetyls); 2.52(3"--N--CH₃); 4.80 (H-4'); 4.92 (H-1", J_(1"),2" = 4.0 Hz); 5.4 (H-1',J_(1'),2' = 2.5 Hz); mass spectrum: (M⁺·) m/e 601, (M + 1)⁺ m/e 602;also m/e 402, 392, 374, 160; 471, 453, 443, 425, 211; and 261. Analysiscalculated for: C₂₇ H₄₇ N₅ O₁₀ ·1.5H₂ O: C, 51.58; H, 8.02; N, 11.14%.Found: C, 51.47; H, 8.89; N, 10.91%.

B. 1-N-Ethylsisomicin

Add 3,2',6'-tri-N-acetyl-1-N-ethylsisomicin (0.1 gm.) to 10 ml. of 1 Nsodium hydroxide and heat the solution under an atmosphere of nitrogenat reflux temperature until thin layer chromatographic analysis of analiquot on silica gel using a 2:1:0.35 mixture ofchloroform:methanol:ammonium hydroxide as the developing systemindicates essential completion of de-N-acetylation (about 48 hours).Cool the reaction solution, add water until the volume is at about 50ml., stir with Amberlite IRC-50 ion exchange resin in the proton form(which has been washed with water) until the pH is at about 5.5. Removethe resin by filtration, wash with water and stir the resin with 100 ml.of 7% aqueous ammonium hydroxide for about 30 minutes, decant thesupernatant solution, and repeat the foregoing procedure twice using 50ml. of 7% aqueous ammonium hydroxide each time then remove the resin byfiltration. Combine the ammonium hydroxide filtrate and decantations,concentrate in vacuo and extract the resultant residue with methanol.Combine the methanol extracts and concentrate in vacuo, dissolve theresultant residue in a solvent mixture consisting ofchloroform:methanol:ammonium hydroxide (2:1:0.35) and pass the solutionthrough a column of alumina (8 gms. 0.8 cm. × 30 cm. column), elutingwith the same chloroform:methanol:ammonium hydroxide solvent mixture.Combine the like eluates as determined by thin layer chromatography andevaporate the combined eluates containing 1-N-ethylsisomicin andlyophilize the resultant aqueous mixture to a residue comprising1-N-ethylsisomicin (71 mg.) (90% yield).

C. Conversion of Sisomicin to 1-N-Ethylsisomicin Without Purification ofthe Intermediates (1) 3,2',6'-Tri-N-Acetylsisomicin

Add cupric acetate hydrate (3 gms., 15 mmol) to a stirred solution ofsisomicin (0.447 gms., 1 mmol) in water (5.33 ml.) and dimethylformamide(18 ml.). Stir at room temperature for 30 minutes, then add dropwise atthe rate of about a drop every three seconds 3.1 ml. of a 1 molarsolution of acetic anhydride in dimethylformamide (3.1 mmol). Stir anadditional 30 minutes, then add 10 ml. of water and bubble hydrogensulfide through the solution. Stir the reaction mixture for 30 minutes,then filter the precipitated cupric sulfide through a pad of Celite,wash the resultant cupric sulfide residue with water, and bring thecombined filtrate to pH 9 by stirring with Amberlite IRA-401S ionexchange resin in the hydroxide cycle. Remove the resin by filtration,wash with water and concentrate the combined filtrate and washings to aresidue comprising 3,2',6'-tri-N-acetylsisomicin.

(2) 3,2',6'-Tri-N-Acetyl-1-N-Ethylsisomicin

Dissolve the product of Example 16C(1) in 10 ml. of water and add 0.1 Nhydrochloric acid until the solution is at a pH of about 2.7. Cool thereaction mixture to about 3° C. and add 0.73 ml. of 1 molar solution ofacetaldehyde in tetrahydrofuran (0.73 mmol), then add dropwise asolution of 0.55 gms. of sodium cyanoborohydride in 0.7 ml. of water.Stir for one hour maintaining the pH of the solution at about 2.7 by theaddition of 0.1 N hydrochloric acid, then add 0.15 ml. of a 1 molarsolution of acetaldehyde in tetrahydrofuran (0.15 mmol) followed by asolution of 12 mg. of sodium cyanoborohydride in a few drops of water,again maintaining the pH at about 2.7. Repeat the foregoing proceduretwice using each time 0.7 ml. of a 1 molar solution of acetaldehyde intetrahydrofuran and 3 mg. of sodium cyanoborohydride. Stir at roomtemperature overnight, then bring the solution to a pH of about 9 with 1N sodium hydroxide. Concentrate the solution in vacuo to a residuecomprising 3,2',6'-tri-N-acetyl-1-N-ethylsisomicin.

(3) 1-N-Ethylsisomicin

Dissolve the 3,2',6'-tri-N-acetyl-1-N-ethylsisomicin obtained in Example16C(2) in 60 ml. of 1 N sodium hydroxide and heat at reflux temperatureunder an atmosphere of nitrogen for 48 hours. Cool and bring thereaction mixture to a pH of about 5.5 by adding Amberlite IRC-50 ionexchange resin in the proton form. Remove the resin by filtration andwash with water. Stir the resin with 100 ml. of 7% aqueous ammoniumhydroxide solution for 30 minutes, decant the solution and repeat theforegoing procedure twice using 100 ml. of 7% ammonium hydroxide eachtime, then filter. Combine the ammonium hydroxide filtrate with thedecantations and evaporate in vacuo, and chromatograph the resultantresidue on a column of silica gel (25 gms.) eluting with a solutionmixture of chloroform:methanol:ammonium hydroxide (2:1:0.34). Combinethe like fractions as determined by thin layer chromatography on silicagel using the same solvent system and concentrate in vacuo the combinedeluates containing 1-N-ethylsisomicin and lyophilize the resultantaqueous mixture to a residue of 1-N-ethylsisomicin, yield 0.233 gms.(49% theory).

EXAMPLE 17 CONVERSION OF 3,2',6'-TRI-N-ACETYLAMINOGLYCOSIDES TO1-N-ETHYLAMINOGLYCOSIDES A. 3,2',6'-Tri-N-Acetyl-1-N-Ethylverdamicin

Dissolve 3,2',6'-tri-N-acetylverdamicin (880 mg., 1.5 mmol) in water (15ml.) and adjust the pH to 2.7 using 0.1 N hydrochloric acid(approximately 28 ml.). Cool in an ice bath to 8°-10° C., then whilestirring add aqueous acetaldehyde (4.1 ml. of 0.73 molar acetaldehyde)in water (2 equivalents). Stir for 5 minutes at 8° C., then add dropwisea solution of sodium cyanoborohydride (182 mg., 3 mmol, 2 equivalents)in 2.5 ml. of water, adjusting the pH of the solution to 2.7 with 0.1 Nhydrochloric acid. Continue maintaining the pH at about 2.7 by additionof 0.1 N hydrochloric acid until the reaction is complete as determinedby thin layer chromatography. Allow the reaction mixture to stand atroom temperature for 18 hours, then freeze dry the reaction mixture andchromatograph the resultant residue on silica gel (90 gm.) in a 1.5 ×126 cm. column eluting with chloroform:methanol:ammonium hydroxide (28%)(30:10:1). Combine the like fractions containing3,2',6'-tri-N-acetyl-1-N-ethylverdamicin as determined by thin layerchromatography. Evaporate the combined eluates in vacuo to a residue of3,2',6'-tri-N-acetyl-1-N-ethylverdamicin, yield 550 mg. (0.84 mmol, 56%theory); mass spectrum: m/e 485, 467, 457, 439, 420, 402, 392, 374; 261,243, 233, 215, 183.

B. 1-N-Ethylverdamicin

Treat 3,2',6'-tri-N-acetyl-1-N-ethylverdamicin (200 mg., 0.304 mmol)with 1 N sodium hydroxide (20 ml.) at reflux temperature under anatmosphere of nitrogen, then isolate and purify the resulting product ina manner similar to that described in Example 16B to obtain1-N-ethylverdamicin, yield 88 mg. (59% theory).

C. Conversion of 3,2',6'-Tri-N-Acetylaminoglycosides to theCorresponding 1-N-Ethyl Derivatives Thereof

In the procedure of Example 16A instead of 3,2',6'-tri-N-acetylsisomicinutilize as starting compounds each of the3,2',6'-tri-N-acetylaminoglycoside derivatives prepared in above Example3A. Isolate and purify each of the resultant products in the mannerdescribed to obtain the corresponding 1-N-ethyl derivative of each ofthe 3,2',6'-tri-N-acetylaminoglycosides.

D. 1-N-Ethylaminoglycosides

In a manner similar to that described in Example 16B treat each of the1-N-ethyl-3,2',6'-tri-N-acetylaminoglycoside derivatives prepared inabove Example 4B with sodium hydroxide followed by isolation andpurification of each of the resultant products in a manner similar tothat described to obtain the corresponding unprotected1-N-ethylaminoglycosides.

EXAMPLE 18 CONVERSION OF 3,6'-DI-N-SUBSTITUTED AMINOGLYCOSIDES TO THECORRESPONDING 3,6'-DI-N-UNSUBSTITUTED-1-N-ALKYLAMINOGLYCOSIDES A.Conversion of 3,6'-Di-N-Tert.-Butoxycarbonylgentamicin B to1-N-Ethylgentamicin B

To a stirred solution of 3,6'-di-N-tert.-butoxycarbonylgentamicin B(1.36 gms.) in water (15 ml.) add a 1 molar solution of acetaldehyde intetrahydrofuran (2 ml.). Adjust the pH of the solution to about 4.9 with0.1 N hydrochloric acid. Add sodium cyanoborohydride (0.2 gm.) andperiodically adjust the pH to about 4.9. Stir for 5 hours at roomtemperature, then evaporate the solution to dryness, add trifluoroaceticacid (10 ml.) to the resultant residue and let stand for 5 minutes. Pourthe reaction solution into ether, isolate the resultant precipitate byfiltration and wash with ether. Chromatograph the precipitate on 100 gm.of silica gel using chloroform:methanol:ammonium hydroxide (3:4:2) asthe solvent mixture. Combine the like fractions containing1-N-ethylgentamicin B as determined by thin layer chromatography,evaporate in vacuo, dissolve the resultant residue in water andlyophilize; yield 40% theory; [α]_(D) ²⁶ + 126.2° (c, 1 in water); pmr(ppm) (D₂ O): δ 5.55 (H-1', J_(1'),2' = 3.0 Hz); 5.05 (H-1", J_(1"),2" =4 Hz); 2.9 (3"--N--CH₃); 1.05-1.5 (2 C--CH₃); mass spectrum: [M + 1]⁺511; also m/e 380, 352, 334; 378, 350, 332; 219, 191, 173. AnalysisCalculated for: C₂₁ H₄₂ O₁₀ N₄.2CO₂ 3H₂ O; C, 42.33; H, 7.41; N, 8.58%.Found: C, 42.37; H, 7.43; N, 8.81%.

B. Conversion of 3,6'-Di-N-Acetylaminoglycosides to the Corresponding1-N-Ethyl Derivatives Thereof

(1) In the procedure of Example 16A, instead of3,2',6'-tri-N-acetylsisomicin, utilize as starting compounds each of the3,6'-di-N-acetylaminoglycoside derivatives prepared in above Example 5A.Isolate and purify each of the resultant products in the mannerdescribed to obtain, respectively,

1. 3,6'-di-N-acetyl-1-N-ethylkanamycin A,

2. 3,6'-di-N-acetyl-1-N-ethylgentamicin B,

3. 3,6'-di-N-acetyl-1-N-ethylgentamicin B₁,

4. 3,6'-di-N-acetyl-1-N-ethylgentamicin B₂,

5. 3,6'-di-N-acetyl-1-N-ethylgentamicin A₃,

6. 3,6'-di-N-acetyl-1-N-ethyl-6'-N-methylkanamycin A.

(2) in a manner similar to that described in Example 16B, treat each ofthe 3,6'-di-N-acetyl-1-N-ethylaminoglycoside derivatives prepared inabove Example 18B with sodium hydroxide followed by isolation andpurification of each of the resultant products in a manner similar tothat described, to obtain the corresponding unprotected1-N-ethylaminoglycosides.

EXAMPLE 19 CONVERSION OF 3,2'-DI-N-ACETYLAMINOGLYCOSIDES TO1-N-ETHYLAMINOGLYCOSIDES A. 3,2'-Di-N-Acetyl-1-N-Ethylaminoglycosides

Treat each of the 3,2'-di-N-acetylaminoglycosides prepared in Example 6Ain a manner similar to that described in Example 16A and isolate andpurify each of the resultant products in a manner similar to thatdescribed to obtain, respectively,

1. 3,2'-di-N-acetyl-1-N-ethylkanamycin C,

2. 3,2'-di-N-acetyl-1-N-ethyl-Antibiotic G-418,

3. 3,2'-di-N-acetyl-1-N-ethylgentamicin A,

4. 3,2'-di-N-acetyl-1-N-ethylgentamicin A₁,

5. 3,2'-di-N-acetyl-1-N-ethylgentamicin X₂.

B. 1-N-Ethylaminoglycosides

Treat each of the 3,2'-di-N-acetyl-1-N-ethylaminoglycosides of Example19A with sodium hydroxide in the manner described in Example 16Bfollowed by isolation and purification of each of the resultant productsin a manner similar to that described to obtain the correspondingunprotected 1-N-ethylaminoglycosides.

EXAMPLE 20 CONVERSION OF 3,6'-DI-N-ACETYLSISOMICIN TO1,2'-DI-N-ETHYLSISOMICIN A. 3,6'-Di-N-Acetyl-1,2'-Di-N-Ethylsisomicin

In a manner similar to that described in Example 16A treat3,6'-di-N-acetylsisomicin with about 2 molar equivalents of acetaldehydein tetrahydrofuran followed by the dropwise addition of about 2 molarequivalents of sodium cyanoborohydride. Isolate and purify the resultantproduct in a manner similar to that described in Example 16A to obtain3,6'-di-N-acetyl-1,2'-di-N-ethylsisomicin.

B. 1,2'-Di-N-Ethylsisomicin

In a manner similar to that described in Example 16B treat3,6'-di-N-acetyl-1,2'-di-N-ethylsisomicin with sodium hydroxide under anatmosphere of nitrogen. Isolate and purify the resultant product in amanner similar to that described to obtain 1,2'-di-N-ethylsisomicin.

PREPARATION OF 1-N-(AMINOHYDROXYALKANOYLAMINOGLYCOSIDES USINGSELECTIVELY BLOCKED INTERMEDIATES EXAMPLE 21 CONVERSION OF3,6'-DI-N-TERT.-BUTOXYCARBONYLGENTAMICIN B TO1-N-(γ-AMINO-α-HYDROXYBUTYRYL)GENTAMICIN B A.1-N-(S-γ-amino-α-hydroxybutyryl)gentamicin B

(1) To a solution of 68.2 mg. (0.1 mmol) of3,6'-di-N-tert.-butoxycarbonylgentamicin B (purified product of Example11F) in water (1 ml.), add methanol (1 ml.), then add dropwise withstirring a solution (0.25 ml.) ofN-(S-γ-benzyloxycarbonylamino-α-hydroxybutyryloxy)succinimide (0.1 mmol)in dimethylformamide (0.4 molar solution). Stir for 1 hour, then addadditional N-(S-γ-benzyloxycarbonylamino-α-hydroxybutyryloxy)succinimide(0.125 ml.). Continue stirring the reaction mixture at room temperaturefor 18 hours, then concentrate in vacuo and dissolve the resultantresidue in methanol (4 ml.) and water (16 ml.). Add 5%palladium-on-carbon catalyst (0.05 gm.) and hydrogenate at 55 psiovernight. Remove the solids by filtration through a pad of Celite, washthe Celite pad with water and concentrate the combined filtrates andwashings to a small volume and lyophilize. Dissolve the resultantresidue in trifluoroacetic acid (0.5 ml.) and set aside for 3 minutes.Pour the solution into excess ether, collect the precipitate byfiltration, wash with ether and dry to give1-N-(S-γ-amino-α-hydroxybutyryl)gentamicin B as the trifluoroacetatesalt. Obtain the free base by dissolving the foregoing salt in water andstirring the solution with Amberlite IRA-401S (OH.sup.⊖). ion exchangeresin. Filter and lyophilize the filtrate to obtain1-N-(S-γ-amino-α-hydroxybutyryl)gentamicin B, yield 49.7 mg. (83%theory).

(2) From Crude 3,6'-Di-N-Tert.-Butoxycarbonylgentamicin B

To the 50 ml. solution comprising3,6'-di-N-tert.-butoxycarbonylgentamicin B prepared in Example 11F, add50 ml. of methanol. To this solution add dropwise with stirring 10 ml.of a 1 molar solution of1-N-(S-γ-benzyloxycarbonylamino-α-hydroxybutyryloxy)succinimide indimethylformamide. Stir for 1 hour and monitor the reaction by thinlayer chromatography using chloroform:methanol:ammonium hydroxide(2:1:0.35). Add an additional 5 ml. of the 1 molar solution of1-N-(S-γ-benzyloxycarbonylamino-α-hydroxybutyryloxy)succinimide and stirthe reaction mixture for 10 hours. Concentrate the solution in vacuo andchromatograph the resultant residue on 300 gm. of silica gel usingchloroform:methanol:ammonium hydroxide (30:10:0.5) as the elutingsolvent. Combine the like fractions containing the1-N-(S-γ-benzyloxycarbonylamino-α-hydroxybutyryl)-3,6'-di-N-tert.-butoxycarbonylgentamicinB and evaporate to a residue. Dissolve the residue in water (50 ml.) andmethanol (18 ml.) and hydrogenate in the presence of 5%palladium-on-carbon (600 mg.) at 55 psi for 10 hours. Remove thecatalyst by filtration through a pad of Celite and wash the residue withwater. Combine the filtrate and washings and evaporate in vacuo toremove the methanol. Wash the resulting aqueous layer 3 times withchloroform (25 ml.) and concentrate the aqueous solution in vacuo anddry the resultant residue over phosphorous pentoxide in vacuo. Dissolvethe resultant residue in a minimum amount of trifluoroacetic acid andset the solution aside for 3 minutes. Add ether and isolate theresultant precipitate by filtration. Wash the precipitate with ether anddry. Dissolve the solid in 30 ml. of water and bring the solution toabout pH 8.5 by stirring with Amberlite IRA-401S (OH.sup.⊖) ion exchangeresin. Remove the solid by filtration, wash with water, combine thefiltrate and washings and lyophilize to give1-N-(S-γ-amino-α-hydroxybutyryl)gentamicin B.

Alternatively, add sulfuric acid to bring the foregoing aqueous solutionto about pH 4.5 and lyophilize to obtain the sulfate salt of1-N-(S-γ-amino-α-hydroxybutyryl)gentamicin B; pmr (ppm) (D₂ O): δ 1.3(4"--C--CH₃); 2.9 (3"--N--CH₃); 4.28 (H-2"', J = 9 Hz and 4 Hz); 5.16(H-1", J_(1"),2" = 3.5 Hz); 6.12 (H-1', J_(1'),2' = 3.0 Hz).

B. 1-N-(R-γ-Amino-α-hydroxybutyryl)gentamicin B

(1) To a solution of 3,6'-di-N-tert.-butoxycarbonylgentamicin B (3.41gms., 5 mmol) in methanol (25 ml.) and water (25 ml.) add with stirringa solution ofN-(R-γ-N-benzyloxycarbonylamino-α-hydroxybutyryloxy)succinimide (1.8gms., 5 mmol) in dimethylformamide (10 ml.). Stir the reaction mixturefor 6 hours, concentrate in vacuo and chromatograph the resultantresidue on silica gel (60-200 mesh, 300 gms.) using a solvent systemcomprising chloroform:methanol:ammonium hydroxide (30:10:0.25). Combinethe like fractions containing the desired product as determined by thinlayer chromatography, concentrate in vacuo and rechromatograph theresidue on the same column using the same solvent system. Combine thelike rechromatographed fractions and evaporate in vacuo to a residuecomprising1-N-(R-γ-N-benzyloxycarbonylamino-α-hydroxybutyryloxy)-3,6'-di-N-tert.-butoxycarbonylgentamicinB.

(2) Dissolve the product of Example 21B(1) in 50% aqueous methanol (20ml.) and hydrogenate over 5% palladium-on-carbon catalyst (0.2 gms.) atroom temperature and 50 psi for 24 hours. Remove the catalyst byfiltration through a pad of Celite, wash the water and evaporate thecombined filtrates in vacuo. Dissolve the resultant residue intrifluoroacetic acid (25 ml.) and set aside for 3 minutes. Add ether andisolate the resultant precipitate comprising the trifluoroacetic acidaddition salt of 1-N-(R-γ-amino-α-hydroxybutyryl)gentamicin B, wash withether and dry. Dissolve the dry precipitate in water (10 ml.) and stirwith Amberlite IRA-401S (OH.sup.⊖) ion exchange resin, sufficient toplace the pH to 9.5. Remove the resin by filtration, wash with water andlyophilize the combined filtrates to a residue comprising pure1-N-(R-γ-amino-α-hydroxybutyryl)-gentamicin B, yield 0.6 gms.; [α]_(D)²⁶ + 118.1° (c, 1 in water). pmr (ppm) (D₂ O): δ 1.23 (4"--C--CH₃); 2.66(3"--N--CH₃); 4.2 (H-2''', J=5Hz and 3Hz); 5.03 (H-1", J=4Hz); 5.4(H-1', J=3.5 Hz). Analysis calculated for: C₂₃ H₄₅ O₁₂ N₅.3CO₂.4H₂ O; C,39.64; H, 6.78; N, 8.89%. Found: C, 39.88; H, 6.58; N, 9.28%.

EXAMPLE 22 CONVERSION OF 3,6'-DI-N-TERT.-BUTOXYCARBONYLGENTAMICIN B TO1-N-(S-β-AMINO-α-HYDROXYPROPIONYL)GENTAMICIN B A.1-N-(S-β-N-Benzyloxycarbonylamino-α-Hydroxypropionyl)-3,6'-Di-N-Tert.-ButoxycarbonylgentamicinB

Add to a stirring solution of 3,6'-di-N-tert.-butoxycarbonylgentamicin B(11.28 g., 16.4 mmol) in methanol (100 ml.) and water (100 ml.), asolution ofN-(S-β-benzyloxycarbonylamino-α-hydroxypropionyloxy)succinimide (10 g.,30 mmol) in 40 ml. of N,N-dimethylformamide over a fifteen minuteperiod. After a period of two hours, evaporate off the solvents in vacuoat 50° C. Chromatograph the resultant residue on 500 g. of silica gelusing a 30:10:0.5 ratio of chloroform:methanol:concentrated ammoniumhydroxide to obtain 13.22 g. of1-N-(S-β-benzyloxycarbonylamino-α-hydroxypropionyl)-3,6'-di-N-tert.-butoxycarbonylgentamicinB.

1-N-(S-β-amino-α-hydroxypropionyl)gentamicin B

Hydrogenate the product of Example 22 at 50 psi at room temperature in amixture of water (730 ml. ) and methanol (240 ml.) in the presence of0.7 g, 5% palladium-on-carbon catalyst for 24 hours. Remove the catalystby filtration through a pad of Celite and wash with water. Concentratethe combined filtrates and dry the residue thoroughly. Dissolve theproduct in trifluoroacetic acid (80 ml.) and set aside for 3 minutes.Pour the solution into excess ether (2 l.) to precipitate thetrifluoroacetic acid salt of pure1-N-(S-β-amino-α-hydroxypropionyl)gentamicin B. Isolate by filtration,wash with ether and dry. Treat the product with Amberlite IRA-401S(OH.sup.⊖) resin in water to pH 9, filter the solution, wash the resinand lyophilize the combined solution to give1-N-(S-β-amino-α-hydroxypropionyl)gentamicin B; [α]_(D) ²⁵ + 112.5° (c,0.4 in water); pmr (ppm) (D₂ O): δ 1.15 (4"--C--CH₃); 1.36 (H-2ax, J₁,2= J₂,3 = J_(1ax),1eq = 12.5 Hz); 1.90 (H-2eq); 2.45 (3"--N--CH₃); 4.06(H-5"eq, J_(5"eq),ax = 13.0 Hz); 4.13 (H-2''', J_(2'''),3''' = ˜ 4.0 and7.0 Hz); 5.05 (H-1", J_(1"),2" = 4.0 Hz); 5.32 (H-1", J_(1"),2" = 3.0Hz). Yield = near quantitative.

C. Carry out the procedure of Examples 22A and 22B utilizingN-(S-δ-benzyloxycarbonylamino-α-hydroxyvaleryloxy)succinimide instead ofthe succinimide reagent specified in Example 22A, whereby is obtained1-N-(S-δ-amino-α-hydroxyvaleryl)gentamicin B.

EXAMPLE 23 CONVERSION OF 3,6'-DI-N-BENZYLOXYCARBONYLAMINOGLYCOSIDES TO1-N-(S-γ-AMINO-α-HYDROXYBUTYRYL)AMINOGLYCOSIDES A. Preparation of1-N-(S-γ-Amino-α-Hydroxybutyryl)kanamycin A

(1) To a stirred solution of 3,6'-di-N-benzyloxycarbonylkanamycin A(1.54 gms., 2 mmol) in tetrahydrofuran (20 ml.) and water (20 ml.) adddropwise a solution ofN-(S-γ-benzyloxycarbonylamino-α-hydroxybutyryloxy)succinimide (1.163gms., 3 mmol) in 2 ml. of N,N-dimethylformamide. Stir for two hours,then concentrate in vacuo and chromatograph the resultant syrupy residueon silica gel (150 gms.) eluting with chloroform:methanol:concentratedammonium hydroxide (2:1:0.35) solvent mixture. Combine the likefractions containing the desired product as determined by thin layerchromatography and evaporate the combined eluates in vacuo to a residuecomprising1-N-(S-γ-benzyloxycarbonylamino-α-hydroxybutyryl)-3,6'-di-N-benzyloxycarbonylkanamycinA; yield 1.3 gm.

(2) Hydrogenate the product of Example 23A(1) in aqueous methanol (1:1)(20 ml.) in the presence of 5% palladium-on-charcoal (0.10 gms.) for 3hours. Separate the catalyst by filtration and evaporate the resultantfiltrate in vacuo to a residue comprising1-N-(S-γ-amino-α-hydroxybutyryl)kanamycin A in quantitative yields.

B. In the procedure of Example 23A, substituteN-(S-β-benzyloxycarbonylamino-α-hydroxypropionyloxy)succinimide forN-(S-γ-benzyloxycarbonylamino-α-hydroxybutyryloxy)succinimide to obtain1-N-(S-β-amino-α-hydroxypropionyl)kanamycin A in an overall yield ofgreater than 50% theory.

C. Treat each of the 3,6'-di-N-benzyloxycarbonylaminoglycoside productsof Example 11E in a manner similar to that described in above Example23A to obtain, respectively,

1. 1-N-(S-γ-amino-α-hydroxybutyryl)gentamicin B₁,

2. 1-n-(s-γ-amino-α-hydroxybutyryl)gentamicin A₃,

3. 1-n-(s-γ-amino-α-hydroxybutyryl)-6'-N-methylkanamycin A.

EXAMPLE 24 CONVERSION OF 3,6'-DI-N-BENZYLOXYCARBONYLGENTAMICIN B TO1-N-(S-β-AMINO-α-HYDROXYPROPIONYL)GENTAMICIN B AND TO1-N-(R-β-AMINO-α-HYDROXYPROPIONYL)GENTAMICIN B A.1-N-(S-β-Amino-α-Hydroxypropionyl)gentamicin B

To a solution of 3,6'-di-N-benzyloxycarbonylgentamicin B (3.75 gms., 5mmol) in methanol (25 ml.) and water (25 ml.), add with stirring asolution ofN-(S-β-benzyloxycarbonylamino-α-hydroxypropionyloxy)succinimide (2.18gms., 6.5 mmol) in dimethylformamide (12 ml.). Stir the reaction mixturefor 2 hours, concentrate in vacuo, and dissolve the resultant residue inwater (75 ml.) and methanol (75 ml.). Hydrogenate in the presence of 5%palladium on charcoal (5 gms.) at 50 psi for 24 hours. Remove thecatalyst by filtration through a pad of Celite and wash with water.Concentrate the combined filtrate and washings and chromatograph theresultant residue on Dowex-1 × 2 ion exchange resin (200 gm., 200-400mesh) in the hydroxide form eluting with water. Combine the likefractions containing 1-N-(S-β-amino-α-hydroxypropionyl)gentamicin B asdetermined by thin layer chromatography on silica gel using chloroform:methanol:ammonium hydroxide (3:4:2) as the solvent system. Concentrateto a small volume, dilute with water and lyophilize to obtain1-N-(S-β-amino-α-hydroxypropionyl)gentamicin B, yield 1.7 gms. (60%theory).

B. 1-N-(R-β-Amino-α-Hydroxypropionyl)gentamicin B

In a manner similar to that described in Example 24A treat3,6'-di-N-benzyloxycarbonylgentamicin B with1-N-(R-β-benzyloxycarbonylamino-α-hydroxypropionyloxy)succinimide andisolate and purify the resultant product in a manner similar to thatdescribed to obtain 1-N-(R-β-amino-α-hydroxypropionyl)-gentamicin B.

C. Conversion of 3,6-Di-N-Benzyloxycarbonylgentamicin B to1-N-(S-β-Amino-α-Hydroxypropionyl)gentamicin B and1-N-(R-β-Amino-α-Hydroxypropionyl)gentamicin B Using Racemicβ-N-Benzyloxycarbonylamino-α-Hydroxypropionic Acid

(1) To a stirred solution of 3,6'-di-N-benzyloxycarbonylgentamicin B (1gm., 1.33 mmol) in methanol (5 ml.) and water (5 ml.) add a solution ofracemic N-(β-benzyloxycarbonylamino-α-hydrxyopropionyloxy)succinimide(0.674 gms., 0.2 mmol) in dimethylformamide (2 ml.). Stir for 2 hours,then evaporate the solvent in vacuo. Chromatograph the resultant residueon silica gel (60-100 mesh, 100 gms.) eluting with a mixture ofchloroform: methanol:ammonium hydroxide (30:10:1). Monitor the fractionsby thin layer chromatography on silica gel using chloroform:methanol:ammonium hydroxide (2:1:0.35) as solvent system. Combine thelike fractions containing1-N-(S-β-benzyloxycarbonylamino-α-hydroxypropionyl)-3,6'-di-N-benzyloxycarbonylgentamicinB, evaporate in vacuo, and water to the resultant residue and lyophilizefrom water, yield 0.42 gms. (65% theory). Similarly, combine the likefractions containing1-N-(R-β-benzyloxycarbonylamino-α-hydroxypropionyl)gentamicin B,evaporate the solvents in vacuo, add water and lyophilize, yield 0.36gms. (55.8% theory).

(2) Hydrogenate each of the two fractions separately in methanol (5 ml.)and water (5 ml.) in the presence of 5% palladium on charcoal catalystat 55 psi for 24 hours. In each instance remove the catalyst byfiltration through Celite, wash with water and evaporate the combinedfiltrates to the residue. Add water to the residue and lyophilize toobtain pure 1-N-(S-β-amino-α-hydroxypropionyl)gentamicin B and1-N-(R-β-amino-α-hydroxypropionyl)gentamicin B, respectively, in nearquantitative yields.

EXAMPLE 25 CONVERSION OF 3,6'-DI-N-BENZYLOXYCARBONYLGENTAMICIN B TO1-N-(S-γ-AMINO-α-HYDROXYBUTYRYL)GENTAMICIN B AND1-N-(R-γ-AMINO-α-HYDROXYBUTYRYL)GENTAMICIN B USING RACEMICN-γ-BENZYLOXYCARBONYL-α-HYDROXYBUTYRIC ACID

In a manner similar to that described in Example 24C treat3,6'-di-N-benzyloxycarbonylgentamicin B in aqueous methanol with asolution of racemicN-γ-benzyloxycarbonylamino-α-hydroxybutyryloxy)succinimide indimethylformamide, then evaporate the reaction mixture in vacuo to aresidue comprising1-N-(R,S-γ-benzyloxycarbonylamino-α-hydroxybutyryl)-3,6'-di-N-benzyloxycarbonylgentamicinB. Chromatograph on silica gel in a manner similar to that described inExample 24C and combine the like fractions as determined by thin layerchromatography and evaporate each of the combined fractions in vacuo toa residue comprising1N-(S-γ-benzyloxycarbonylamino-α-hydroxybutyryl)-3,6'-di-N-benzyloxycarbonylgentamicinB and1-N-(R-γ-benzyloxycarbonylamino-α-hydroxybutyryl)-3,6'-di-N-benzyloxycarbonylgentamicinB, respectively. Hydrogenate each of the two fractions in the presenceof 5% palladium-on-charcoal catalyst similar to that described inExample 24C(2) to obtain 1-N-(S-γ-amino-α-hydroxybutyryl)gentamicin Band 1-N(R-γ-amino-α-hydroxybutyryl)gentamicin B, respectively.

We claim:
 1. The process for selectively blocking amino groups with anacyl blocking group, Y, in a polyamino-organic compound, at least one ofsaid amino groups having an available neighboring hydroxyl group, and atleast one of said amino groups being either devoid of, or stericallyless available to, an available neighboring hydroxyl group;whichcomprises the reaction of said polyamino-organic compound in an inertorganic solvent, with a salt of a divalent transition metal cationselected from the group consisting of copper (II), nickel (II), cobalt(II) and cadmium (II) or with mixtures thereof, whereby is formed acomplex of said polyamino-organic compound between said transition metalsalt and said available neighboring amino and hydroxyl group pairs;followed by the reaction in situ of the resulting polyamino-organiccompound-transition metal salt complex with an amine blocking reagenthaving an acyl blocking group Y; thence reaction of the resultingpolyamino-organic compound-transition metal salt complex having acylblocking groups, Y, on non-complexed amino groups, with a transitionmetal precipitating reagent or with ammonium hydroxide, whereby saidtransition metal cation is removed.
 2. The process of claim 1 whereinsaid transition metal precipitating reagent is a member selected fromthe group consisting of acetyl-acetone, dioxime of dimethylglyoxal, andhydrogen sulfide.
 3. The process of claim 1 wherein said dilventtransition metal cation is a member selected from the group consistingof copper (II), nickel (II) and cobalt (II), and said transition metalcation is removed from said polyamino-organic compound salt complexhaving acyl blocking groups, Y, by reaction with hydrogen sulfide orwith ammonium hydroxide.
 4. The process of claim 1 wherein said salt ofsaid divalent transition metal cation is a member selected from thegroup consisting of cupric acetate, nickel (II) acetate and cobalt (II)acetate, the total molar quantity of said transition metal salt being atleast about equal to the number of available neighboring amino andhydroxyl group pairs, and the molar quantity of said acyl blockingreagent being about equal to the number of amino groups devoid of atransition metal salt complex to be selectively blocked, and whereinsaid transition metal cation is removed from said resulting transitionmetal salt complex of said polyamino-organic compound having acylblocking groups, Y, by reaction with hydrogen sulfide or with ammoniumhydroxide.
 5. The process of claim 4 wherein said polyamino-organiccompound is an aminocyclitol-aminoglycoside.
 6. The process of claim 5wherein said aminocyclitol-aminoglycoside is a4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol which has availablehydroxyl groups neighboring to every amino group except those at the 3,2' and 6' positions; which comprises the reaction of said4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol in an inert aprotic organicsolvent with a salt of a divalent transition metal cation selected fromthe group consisting of copper (II), nickel (II), cobalt (II) or with amixture thereof, the total molar quantity of said transition metal saidor mixture thereof being at least about equal to the molar quantity ofsaid 4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol times the number ofavailable neighboring hydroxyl and amino group pairs; followed by thereaction in situ of the resulting4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol-transition metal saltcomplex with about three moles of an acylating reagent having an acylgroup, Y, followed by reaction of the resulting3,2',6'-tri-N-Y-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol-transitionmetal salt complex with hydrogen sulfide or ammonium hydroxide wherebyis produced the corresponding3,2',6'-tri-N-Y-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol.
 7. Theprocess of claim 6 wherein said4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol is a member selected fromthe group consisting of sisomicin, verdamicin, tobramycin, gentamicinC₁, gentamicin C_(1a), gentamicin C₂, gentamicin C_(2a), gentamicinC_(2b), Antibiotic 66-40B, Antibiotic 66-40D, Antibiotic JI-20A,Antibiotic JI-20B, Antibiotic G-52,the 5-epi- and 5-epi-azido-5-deoxyanalogs of the foregoing; kanamycin B, 3',4'-dideoxykanamycin B,nebramycin factor 4, nebramycin factor5',3',4'-dideoxy-3',4'-dehydrokanamycin B,3',4'-dideoxy-6'-N-methylkanamycin B and 5-deoxysisomicin, wherein themolar quantity of said transition metal salt or mixture thereof is atleast two times that of said4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol.
 8. The process of claim 7wherein said acylating reagent is acetic anhydride whereby is producedthe corresponding3,2',6'-tri-N-acetyl-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol. 9.The process of claim 8 wherein said4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol is sisomicin whereby isformed 3,2',6'-tri-N-acetylsisomicin.
 10. The process of claim 7 whereinsaid acylating reagent isN-(2,2,2-trichloroethoxycarbonyloxy)succinimide whereby is formed thecorresponding3,2',6'-tri-N-(2,2,2-trichloroethoxycarbonyl)-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol.11. The process of claim 10 wherein said4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol is sisomicin whereby isformed 3,2',6'-tri-N-(2,2,2-trichloroethoxycarbonyl)sisomicin.
 12. Theprocess of claim 5 wherein said aminocyclitol-aminoglycoside is a4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol which has availablehydroxyl groups neighboring to every amino function except those at the3 and 6' positions and is a member selected from the group consisting ofgentamicin B, gentamicin B₁, gentamicin A₃, kanamycin A and6'-N-methylkanamycin A, which comprises the reaction of said4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol in an inert aprotic organicsolvent with a salt of a divalent transition metal cation selected fromthe group consisting of copper (II), nickel (II) and cobalt (II) or witha mixture thereof, the total molar quantity of said salts or mixturethereof being at least two times the molar quantity of said4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol, followed by the reactionin situ of the resulting4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol-transition metal saltcomplex with about two moles of an acylating reagent having an acylgroup, Y; followed by reaction of the resulting3,6'-di-N-Y-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol-transitionmetal salt complex with hydrogen sulfide or ammonium hydroxide, wherebyis produced the corresponding3,6'-di-N-Y-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol.
 13. Theprocess of claim 12 wherein said4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol is a member selected fromthe group consisting of gentamicin B, gentamicin B₁ kanamycin A, andwherein said acylating reagent is a member selected from the groupconsisting of acetic anyhydride, N-tert.-butoxycarbonyloxy-phthalimideand N-benzyloxycarbonyloxyphthalimide, whereby is formed thecorresponding 3,6-di-N-acetyl, 3,6-di-N-tert.-butoxycarbonyl and3,6'-di-N-benzyloxycarbonyl derivatives of a member selected from thegroup consisting of gentamicin B, gentamicin B₁ and kanamycin A.
 14. Theprocess of claim 5 wherein said aminocyclitol-aminoglycoside is a4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol which has availablehydroxyl groups neighboring to every amino function except the 2' andthe 3-amino groups, and is a member selected from the group consistingof kanamycin C, Antibiotic G-418, gentamicins A, A₁, and X₂, whichcomprises the reaction of said4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol is an inert aprotic organicsolvent with a salt of a divalent transition metal cation selected fromthe group consisting of copper (II), of nickel (II), of cobalt (II), anda mixture thereof, the total molar quantity of said salts or mixturethereof being at least about two times the molar quantity of said4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol; followed by the reactionin situ of the resulting 4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitoltransition metal salt complex with about two moles of an acylatingreagent having an acyl group, Y; follwed by reaction of the resulting3,2'-di-N-Y-4,6-di-O-(aminogylcosyl)-1,3-diaminocyclitol-transitionmetal salt complex with hydrogen sulfide or ammonium hydroxide, wherebyis produced the corresponding3,2'-di-N-Y-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol.
 15. Theprocess of claim 5 wherein said aminocyclitol-aminoglycoside is a4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol which has availablehydroxyl groups neighboring to every amino function except those at the3, 2' and 6'positions which comprises the reaction of said4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol in an inert organic solventwith a salt of a divalent transition metal cation selected from thegroup consisting of copper (II), nickel (II), cobalt (II) or with amixture thereof, the total molar quantity of said transition metal saltsor mixtures thereof being at least about two times that of said4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol; followed by the reactionin situ of the resulting4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol-transition metal saltcomplex with about two moles of an acylating reagent having an acylgroup, Y; followed by reaction of the resulting2',6'-di-N-Y-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol-transitionmetal salt complex with hydrogen sulfide or ammonium hydroxide, wherebyis produced the corresponding2',6'-di-N-Y-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol.
 16. Theprocess of claim 7 including the subsequent steps comprising thereaction of an acid addition salt of the3,2',6'-tri-N-acyl-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol therebyformed with about one equivalent of a hydride-donor reducing reagentselected from the group consisting of dialkylaminoborane,tetralkylammonium cycanoborohydride, alkali metal cyanoborohydride andalkali metal borohydride in an inert solvent and with at least oneequivalent of an aldehyde having the formula X'CHO wherein X' is amember selected from the group consisting of hydrogen and a substituentselected from the group consisting of alkyl, cycloalkyl, alkenyl,aralkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, aminohydroxalkyl,alkylaminohydroxyalkyl, dialkyaminoalkyl and dialkylaminohydroxyalkyl,said substituent having up to 7 carbon atoms and when said substituentis substituted by both hydroxyl and amino functions, only one of saidfunctions can be attached to any one carbon; followed by removal of saidacyl protecting groups, Y, and of said acid addition salt, whereby isobtained a 1-N-X derivative of said4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol whereby X is --CH₂ X', X'being as hereinabove defined.
 17. The process of claim 16 wherein saidacylating reagent is acetic anhydride, said aldehyde, X'CHO, isacetaldehyde, said reducing agent is sodium cyanoborohydride, and saidacyl protecting groups, Y, and the acid of said acid addition salt areremoved by the reaction of the thereby formed1-N-ethyl-3,2',6'-tri-N-acetyl-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitolacid addition salt by treatment with base whereby is obtained thecorresponding 1-N-ethyl-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol.18. The process of claim 17 wherein said4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol is sisomicin whereby isobtained 1-N-ethylsisomicin.
 19. The process of claim 13 wherein saidacyl blocking group, Y is tert.-butoxycarbonyl or benzyloxycarbonylincluding the subsequent steps comprising the reaction of the therebyformed 3,6'-di-N-acyl-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol withan acid of the formula HO--X' wherein X' is β-amino-α-hydroxypropionyl,γ-amino-α-hydroxybutyryl, or δ-amino-α-hydroxyvaleryl, said amino groupsbeing protected by an acyl function, Y', in the presence of acarbodiimide, or with a reactive derivative of said acid,followed byremoval of said protecting groups Y and Y' whereby is formed a 1-N-X'derivative of kanamycin A, gentamicin B and gentamicin B₁, X' being ashereinabove defined.
 20. The process of claim 19 wherein said4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol is kanamycin A, andwwherein said reactive derivative of said acid isN-(S-γ-benzyloxycarbonylamino-α-hydroxybutyryloxy)succininimide whichcomprises the reaction of the thereby formed 3,6'-di-N-Y-kanamycin Awith N-(S-γ-benzyloxycarbonylamino-α-hydroxybutyryloxy)succinimidefollowed by removal of said blocking groups, Y, in the resulting1-N-(S-γ-benzyloxycarbonylamino-α-hydroxybutyryl)-3,6'-di-N-Y-kanamycinA to form 1-N-(S-γ-amino-α-hydroxybutyryl)kanamycin A.
 21. The processof claim 19 wherein said 4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol isgentamicin B, and said reactive derivative of said acid is a memberselected from the group consisting ofN-(β-benzyloxycarbonylamino-α-hydroxypropionyloxy)succinimide,N-(γ-benzyloxycarbonylamino-α-hydroxybutyryloxy)succinimide, andN-(δ-benzyloxycarbonylamino-α-hydroxyvaleryloxy)succinimide, whereby isobtained the corresponding member selected from the group consisting of1-N-(β-amino-α-hydroxypropionyl)gentamicin B,1-N-(γ-amino-α-hydroxybutyryl)gentamicin B and1-N-(δ-amino-α-hydroxyvaleryl)gentamicin B.
 22. The process of claim 21wherein said reactive derivative of said acid isN-(S,R-β-benzyloxycarbonylamino-α-hydroxypropionyloxy)succinimide orN-(S,R-γ-benzyloxycarbonylamino-α-hydroxybutyryloxy)succinimide whichcomprises the reaction of the thereby formed 3,6'-di-N-Y-gentamicin Bwith N-(S,R-62-benzyloxycarbonylamino-α-hydroxypropionyloxy)-succinimide orN-(S,R-γ-benzyloxycarbonylamino-α-hydroxybutyryloxy)succinimide followedby separation of the diastereoisomers in the thereby formed1-(N-(S,R-β-benzyloxycarbonylamino-α-hydroxypropionyl)-3,6'-di-N-gentamicinB or1-N-(S,R-γ-benzyloxycarbonylamino-α-hydroxybutyryl)-3,6'-di-N-Y-gentamicinB, respectively, and thence removal of said blocking groups, Y, fromeach of the diastereoisomers thereby separated whereby is obtained amember selected from the group consisting of1-N-(S-β-aino-α-hydroxypropionyl)gentamicin B,1-N-(R-β-amino-α-hydroxypropionyl)gentamicin B,1-N-(S-γ-amino-α-hydroxybutyryl)gentamicin B and1-N-(R-γ-amino-α-hydroxybutyryl)-gentamicin B, respectively.
 23. Theprocess of claim 12 including the subsequent steps comprising thereaction of an acid addition salt of the3,6'-di-N-acyl-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol therebyformed with about one equivalent of a hydride-donor reducing agentselected from the group consisting of dialkylaminoborane,tetraalkylammonium cycanoborohydride, alkali metal cyanoborohydride andalkali metal borohydride in an inert solvent and with at least oneequivalent of an aldehyde having the formula X'CHO wherein X' is amember selected from the group consisting of hydrogen and a substituentselected from the group consisting of alkyl, cycloalkyl, alkenyl,aralkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, aminohydroxyalkyl,alkylaminohydroxyalkyl, dialkylaminoalkyl and dialkylaminohydroxyalkyl,said subsituent having up to 7 carbon atoms and when said substituent issubstituted by both hydroxyl and amino functions, only one of saidfunctions can be attached to any one carbon; followed by removal of saidacyl protecting groups, Y, and of the acid of said acid addition salt,whereby is obtained a 1-N-X derivative of said4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol whereby X is --CH₂ X', X'being as hereinabove defined.
 24. The process of claim 23 wherein saidacylating reagent is acetic anhydride, said aldehyde, X'CHO, isacetaldehyde, said reducing agent is sodium cyanoborohydride, and saidacyl protecting group, Y, and the acid of said acid addition salt of thethereby formed1-N-ethyl-3,6'-di-N-acetyl-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitolis removed by reaction thereof with base whereby is obtained thecorresponding 1-N-ethyl-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol.